Compositions and methods for regulation of gene expression with, and detection of, folinic acid and folates

ABSTRACT

Aptamers that specifically bind to ligands of folinic acid, a folate, and derivatives thereof (which may be referred to herein as ligands) are provided, and compositions and methods of use thereof. The aptamers and switches of the invention provide biological sensing capability for detecting the ligands, and are effective in sensing in vitro and in vivo. By specific sensing of the ligand, the aptamers of the invention provide a means of engineering an inducible gene regulatory system that enables dose-dependent control over gene expression in response to the ligand, in vivo and in vitro.

CROSS-REFERENCE

This application is a continuation and claims benefit of 371 applicationSer. No. 15/502,694, filed Feb. 8, 2017, which claims the benefit of PCTApplication No. PCT/US2015/045120, filed Aug. 13, 2015, which claims thebenefit of U.S. Provisional Application No. 62/037,015, filed Aug. 13,2014, which applications are incorporated herein by reference in theirentirety.

GOVERNMENT SUPPORT

This invention was made with Government support under contractHR0011-11-2-0002 awarded by the Defense Advanced Research ProjectsAgency and under contract GM091298 awarded by the National Institutes ofHealth. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Chemical induction of gene expression is a regulatory tool in nature andin biotechnology for constructing, studying, and engineering biologicalsystems. Inducible promoters and riboswitches found in naturedynamically control metabolism and cellular communication in response tointracellular and environmental signals. Many of these natural systemshave been repurposed in biotechnology. However, these systems arerestricted to a limited set of existing and well-characterizedbiological sensing capabilities; are often constrained for use inparticular organisms; and may be challenging to functionally decouplefrom native cellular regulation. Efforts to expand this natural chemicaldiversity have included significant advances in the engineering ofsynthetic riboswitches, which can incorporate novel RNA sensors, oraptamers, generated de novo through in vitro selection and operatethrough varied RNA regulatory mechanisms. See for example, Chang et al.(2012) Current Opinion in Biotechnology 23(5):679-688.

There is a need, therefore, to develop aptamers for gene regulatorysystems that can control the expression of specific target genes in vivoin response to effector molecules.

SUMMARY OF THE INVENTION

Compositions and methods are provided that relate to aptamers thatspecifically bind to ligands of folinic acid, a folate, and derivativesthereof (which may be referred to herein as ligands). The aptamers andswitches of the invention provide biological sensing capability fordetecting the ligands, and are effective in sensing in vitro and invivo. The aptamers can be coupled to a suitable actuator, e.g. a primingsequence domain, ribozyme, etc. for regulation of gene expression inresponse to the ligand. By specific sensing of the ligand, the aptamersof the invention provide a means of engineering an inducible generegulatory system that enables dose-dependent control over geneexpression in response to the ligand, in vivo and in vitro. Thistechnology provides a means for controlling RNA-based, viral, orcellular therapeutics using an externally administered ligand. It alsoenables diagnostic detection and measurement of the ligand, i.e. folinicacid, folate derivatives, etc. The aptamers also find use as a generalinducible gene expression system for a variety of biotechnologyapplications.

RNA aptamer sensors that bind with high specificity and affinity tofolinic acid were generated de novo through in vitro selection.Exemplary sequences are provided herein for high affinity and highlyselective sensors. The invention also provides the sequences forfunctional microRNA-based switches useful in mammalian cells. Thesensing and regulatory capabilities are applicable for use in anyorganism, including bacteria, fungi, plants, mammalian cells, andviruses. The aptamer sensors can be used independently as an in vitrodiagnostic tool for folinic acid or other folate derivatives. Inaddition, the ligand can be modified to alter or remove certainfunctional groups or to covalently attach other molecules, nucleicacids, proteins, nanoparticles, or drugs of interest, as shown, forexample in Table 6.

A benefit of the aptamers of the invention may be the detection ofligands suitable for clinical use. Folinic acid is FDA-approved andclinically used. The folinic acid (6R)-diastereomer is not naturallypresent in cells, is not biologically active and exhibits high stabilityand low toxicity, making it highly suitable for gene regulation. Theseligand properties enable the developed sensors and gene-regulatorydevices of the invention to be widely used in clinically applicablesystems.

Compositions of the invention include isolated RNA aptamers and DNAsequences encoding such aptamers; and vectors and cells comprising suchRNA and DNA compositions. The RNA aptamers or DNA encoding such aptamersmay be operably joined in an aptamer-regulated device, (also referred toas a gene regulatory system), to an actuator, generally apolynucleotide-based actuator, e.g. including without limitationriboswitches, ribozymes, microRNAs, antisense RNAs, RNAi, CRISPR,splicing, small RNAs, ribosome binding sites, internal ribosome entrysites, etc. Compositions of the invention also include isolated RNAdevices and DNA sequences encoding such devices; and vectors and cellscomprising such devices and encoding sequences.

In addition to a nucleic acid, which is itself functional as anaptamer-regulated device for responding to changes in ligandconcentration, (for example a device comprising an aptamer of theinvention and an actuator), the invention also provides expressionconstructs that include a “coding sequence” which, when transcribed toRNA, produces the aptamer-regulated device. The expression construct mayinclude one or more transcriptional regulatory sequences that regulatetranscription of that sequence in a cell containing the expressionconstruct. The expression construct can be designed to include one ormore actuators in an RNA transcript, such as in the 3′ untranslatedregion (3′-UTR), so as to regulate transcription, stability and/ortranslation of that RNA transcript in a manner dependent on the ligand.For example, the expression construct can include a coding sequence fora polypeptide such that the mRNA transcript includes both thepolypeptide coding sequence as well as one or more of the regulatedactuators. In this way, expression of the polypeptide can be rendereddependent on the ligand to which the aptamer binds. The presentinvention also provides cells that have been engineered to include suchexpression constructs. Still another aspect of the invention relates tomethods for regulating expression of a recombinant gene. Those methodsinclude providing such a cell, and contacting the cell with the ligandin an amount that alters the activity of the actuator, and therefore,the expression of the recombinant gene. In all such embodiments,included those listed below, the folinic acid, a folate, and derivativesthereof can be administered by any convenient means, e.g. oral,parenteral, in culture medium or growth medium, etc. to regulateexpression through the aptamer-regulated device.

Another aspect of the invention provides a cell having a metabolicpathway of one or more reactions, and in which one or more ofaptamer-regulated devices act as control elements on the metabolicpathway by regulating expression of one or more target genes. In suchembodiments, ligand binding to the aptamer causes a change in theactuator between two conformational states, in one of which the actuatorinhibits expression of a target gene and in the other of which theactuator does not inhibit expression of the target gene. In thisembodiment, the metabolic pathway is regulated at least in part by theactivity level of the actuator, and therefore, the level of ligand thatis present. Such embodiments may be used to regulate a metabolic pathwaythat includes at least one reaction mediated by an enzyme, such as wherethe actuator regulates expression of the enzyme.

Another aspect of the invention provides a cell having a metabolicpathway of one or more reactions, and in which one or more of thesubject aptamer-regulated devices act as control elements on themetabolic pathway by inhibiting expression of one or more target genesinto which the device has been engineered so as to be part of the m RNAtranscript of the gene, preferably as part of the 3′-UTR. In suchembodiments, the ligand binding to the aptamer causes a change in theactuator between two conformational states, in one of which the actuatorinhibits expression of the target gene. In this embodiment, themetabolic pathway is regulated at least in part by the activity level ofthe actuator, and therefore, the level of ligand present. Suchembodiments may be used to regulate a metabolic pathway that includes atleast one reaction mediated by an enzyme, such as where the actuatorregulates expression of the enzyme.

Another aspect of the invention provides a method for renderingexpression of a target gene in a cell dependent on the presence orabsence of a ligand, by utilizing a version of the subjectaptamer-regulated device that, in its active form, cleaves a transcriptproduced by transcription of the target gene, and thereby inhibitsexpression of the target gene in a manner dependent on the presence orabsence of the ligand.

Likewise, the aptamer-regulated device of the invention can be used torender expression of a target gene in a cell dependent on the presenceor absence of a ligand. In these embodiments, the cell is engineeredwith an expression construct that includes a coding sequence for thetarget gene, which when transcribed to an mRNA transcript, also includesone or more aptamer-regulated devices in the mRNA. Ligand binding to theaptamer causes a change in the device between two conformational states,in one of which the actuator inhibits expression of the target genepresent with the actuator in the same transcript. In this way, theaptamer-regulated device present in the transcript can regulatetranscription, stability and/or translation of the mRNA in a mannerdependent on the ligand.

In still another embodiment, the present invention provides a method fordetermining the amount of a ligand of the invention in a cell whichexpresses a reporter gene by way of the cell also containing anaptamer-regulated device that changes the reporter gene in a mannerdependent on the level of the ligand of the invention. The method caninclude measuring the amount of expression of the reporter gene, andcorrelating the amount of expression of the reporter gene with theamount of ligand, thereby determining the amount of the ligand in thecell. Exemplary reporter molecules include, without limitation,fluorescent or luminescent reporter proteins such as fluorescentproteins (e.g., green fluorescent protein (GFP), mCherry), luciferase,or aptamers (e.g., Spinach, Spinach2, Broccoli) that specifically bindfluorescent molecules (e.g., 3,5-difluoro-4-hydroxybenzylideneimidazolinone); enzymatic reporters such as alkaline phosphatase; orcolorimetric reporters such as lacZ.

In some specific embodiments, an aptamer of the invention is used toregulate a ribozyme. Various ribozymes find use, including withoutlimitation a cis-acting hammerhead ribozyme. In general a regulatedribozyme includes a ribozyme with at least one aptamer of the inventiondirectly coupled to a ribozyme. Suitable switches are provided herein,for example in Table 5 and Table 7. In certain embodiments the aptamermay bind folinic acid, a folate, or derivatives thereof in a manner thatalters the base-pairing with a transmitter component that is carriedover as a structural change in the ribozyme. In certain embodiments, theaptamer is integrated such that binding of the ligand to the aptamercauses a change with one or more of the loop, the stem or the catalyticcore of the ribozyme, such that the ribozyme undergoes self-cleavage ofa backbone phosphodiester bond at a rate dependent upon the presence orabsence of the ligand. In certain embodiments, the presence of ligandwill increase the rate of self-cleavage relative to the absence ofligand, while in other embodiments the rate of self-cleavage is greaterin the absence of ligand relative to the presence of ligand. In certainpreferred embodiments, the difference in the rate of cleavage is atleast 100 fold, and even more preferably 1000 or 10,000 fold.

Another aspect of the invention provides pharmaceutical preparations andcompositions comprising an aptamer of the present invention, anaptamer-regulated device, an expression construct which, whentranscribed, produces an RNA including the aptamer or aptamer-regulateddevice, and a pharmaceutically acceptable carrier suitable foradministration use to a human or non-human patient. Optionally, thepharmaceutically acceptable carrier is selected from pharmaceuticallyacceptable salts, ester, and salts of such esters. In certain preferredembodiments, the present invention provides a pharmaceutical package orkit comprising the pharmaceutical preparation which includes at leastone aptamer-regulated device and a pharmaceutically acceptable carrier,in association with instructions (written and/or pictorial) foradministering the preparation to a human patient.

In one aspect, the disclosure provides an aptamer that specificallybinds to a ligand of folinic acid, a folate, or a derivative thereof. Insome embodiments, the aptamer is RNA. In some embodiments, the aptameris up to 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides inlength. In some embodiments, the aptamer is an RNA of up to 80nucleotides in length. In some embodiments, the aptamer is an RNA offrom 15 to 80 nucleotides in length. In some embodiments, the ligand isselected from (6S)-folinic acid; (6R)-folinic acid; a diastereomericmixture of (6R)- and (6S)-folinic acid; tetrahydrofolic acid;(6R)-tetrahydrobiopterin; folic acid; dihydrofolic acid;5-formiminotetrahydrofolate; 10-formyltetrahydrofolate;5,10-methenyltetrahydrofolate; 5,10-methylenetetrahydrofolate;levomefolic acid; technetium (99mTc) etarfolatide; tetrahydrofolic acid;vintafolide; methotrexate; pemetrexed; tetrahydrofolic acid;(6R)-tetrahydrobiopterin; (6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid;(6R)-5-methyl-5,6,7,8-tetrahydrofolic acid;(6S)-5-methyl-5,6,7,8-tetrahydrofolic acid;(6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acid;(6R)-5-formyl-5,6,7,8-tetrahydropteroic acid;(6S)-5-formyl-5,6,7,8-tetrahydropteroic acid; a tagged derivativethereof; and any combination thereof. In some embodiments, the ligand isselected from (6S)-folinic acid; (6R)-folinic acid; a diastereomericmixture of (6R)- and (6S)-folinic acid; and a tagged derivative thereof.In some embodiments, the aptamer comprises a nucleotide sequence setforth in any one of FIG. 4, FIG. 13, FIG. 14, or FIG. 18; or a variantthereof. In some embodiments, the aptamer comprises a truncated sequenceof a nucleotide sequence set forth in any one of FIG. 4, FIG. 13, FIG.14, or FIG. 18; or a variant thereof. In some embodiments, the aptamercomprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%, or 100% identity to a truncated sequence. In someembodiments, the aptamer specifically binds (6R)-folinic acid. In someembodiments, the aptamer comprises a sequence having at least 70%identity sequence to FAt8-4, FAt8-11, FAt8-3, FAt6-8, or FAt8-16. Insome embodiments, the aptamer specifically binds (6S)-folinic acid. Insome embodiments, the aptamer comprises a sequence having at least 70%identity sequence to FAt8-18, FAt10-7, or FAt8-14. In some embodiments,the aptamer comprises a sequence or a sequence having at least 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identity to asequence selected from: (i) N₁ N₂ G N₃ U G C G U G G U A C G U U A U A UU C C G N₄ N₅ (SEQ ID NO:129),where N₁ and N₅ are any complementary pairof nucleotides; N₂ and N₄ are any complementary pair of nucleotides; andN₃ is any nucleotide; (ii) N₁ N₂ U G C N₃ U G G U A C G U U A U A U U CR G N₄ N₅ (SEQ ID NO:130), where N₁ and N₅ are any complementary pair ofnucleotides; N₂ and N₄ are any complementary pair of nucleotides; N₃ isany nucleotide; and R is an A or G nucleotide; (iii) C G U C U G G U C AC G A C C N₁ N₂-N₃ N₄ C C C U C G A A A U C A C G A G G G R G A C R A GA Y (SEQ ID NO:131), where N₁ and N₄ are any complementary pair ofnucleotides; N₂ and N₃ are any complementary pair of nucleotides; R isan A or G nucleotide; Y is a C or U nucleotide; and the dash indicatesany intervening sequence of nucleotides or two separate nucleotidestrands; and (iv) N₁ N₂ G G C G A A G A G U C A A A G C A U C C C CN₃-N₄ G G G C C C N₅ N₆ (SEQ ID NO:132), where N₁ and N₆ are anycomplementary pair of nucleotides; N₂ and N₅ are any complementary pairof nucleotides; N₃ and N₄ are any complementary pair of nucleotides; andthe dash indicates any intervening sequence of nucleotides or twoseparate nucleotide strands. In some embodiments, the aptamerspecifically binds to a ligand with a K_(D) of up to 100 nM, 200 nM, 300nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 μM, 2 μM, 3 μM, 4μM, 5 μM. In some embodiments, the aptamer specifically binds to aligand with a K_(D) within the range from 100 μM to 1 μM.

In some embodiments, the aptamer is operably linked to an actuator togenerate an aptamer-regulated device. In some embodiments, the actuatoris a ribozyme, such as a hammerhead ribozyme. In some embodiments, theaptamer-regulated device comprises a sequence set forth in Table 5. Insome embodiments, the actuator is selected from microRNAs, antisenseRNAs, RNAi, CRISPR, splicing, small RNAs, ribosome binding sites,internal ribosome entry sites, aptamers, and any combination thereof.

In one aspect, the disclosure provides an aptamer-regulated device, thedevice comprising an aptamer operably linked to an actuator, wherein theactuator is a hammerhead ribozyme and the aptamer and stem III of thehammerhead ribozyme comprise one or more shared base pairs. In someembodiments, the one or more shared base pairs comprise 2, 3, 4, 5, ormore than 5 shared base pairs. In some embodiments, theaptamer-regulated device comprises a hammerhead ribozyme interveningsequence of nucleotides within the aptamer sequence. In someembodiments, the aptamer-regulated device comprises an aptamer oraptamer-regulated device disclosed herein. In some embodiments, theaptamer-regulated device comprises an aptamer that specifically binds toa ligand selected from theophylline, xanthine, 3-methylxanthine,tetracycline, neomycin, hypoxanthine, tetramethylrosamine,p-aminophenylalanine, biotin, 2,4-dinitrotoluene, hoechst 33342,tryptophan, thiamine pyrophosphate, guanine, adenine, 2-aminopurine,azacytosine, ammeline, PPAO, PPDA, purine, and any combination thereof.

In one aspect, the disclosure provides a polynucleotide encoding anyaptamer or aptamer-regulated device described herein. In someembodiments, the polynucleotide is a DNA sequence. In one aspect, thedisclosure provides a vector comprising any polynucleotide or DNAsequence described herein. In some embodiments, the DNA sequence isoperably linked to a promoter for expression in a cell of interest. Inone aspect, the disclosure provides a cell comprising any vectordescribed herein.

In one aspect, the disclosure provides a method of regulating geneexpression in a cell, the method comprising introducing into the cellany aptamer, DNA sequence, aptamer-regulated device, or vector describedherein, under conditions where an actuator acts on a genetic sequence ofinterest expressed by the cell; and providing the cell with a ligand ofthe aptamer to regulate gene expression. In some embodiments, the cellis in vivo. In some embodiments, the cell is in vitro. In someembodiments, the genetic sequence of interest encodes a reporterprotein. In some embodiments, the genetic sequence of interest encodes atherapeutic protein. In some embodiments, the genetic sequence ofinterest is an RNA-based therapeutic. In some embodiments, the cell is amammalian cell, bacterial cell, fungal cell, algal cell, or plant cell.

In one aspect, the disclosure provides a method of modulating RNAexpression, the method comprising providing an RNA or a DNA sequenceencoding the RNA with a ligand of an aptamer under conditions where anactuator acts on the RNA or the DNA sequence encoding the RNA tomodulate RNA expression, wherein the RNA or the DNA sequence encodingthe RNA comprises any aptamer, DNA sequence, or aptamer-regulated devicedescribed herein. In some embodiments, the providing an RNA or a DNAsequence encoding the RNA with a ligand of an aptamer results in anincrease in RNA expression. In some embodiments, the providing an RNA ora DNA sequence encoding the RNA with a ligand of an aptamer results in adecrease in RNA expression.

In one aspect, the disclosure provides a method of modulating proteinexpression, the method comprising providing an RNA or a DNA sequenceencoding a protein with a ligand of an aptamer under conditions where anactuator acts on the RNA or the DNA sequence encoding the protein tomodulate protein expression, wherein the RNA or the DNA sequenceencoding the protein comprises any aptamer, DNA sequence, oraptamer-regulated device described herein. In some embodiments, theproviding an RNA or a DNA sequence encoding a protein with a ligand ofan aptamer results in an increase in protein expression. In someembodiments, the providing an RNA or a DNA sequence encoding a proteinwith a ligand of an aptamer results in a decrease in protein expression.

In one aspect, the disclosure provides a method of determining ligandconcentration, the method comprising providing any aptamer, DNAsequence, aptamer-regulated device, vector, or cell described hereinwith a ligand of the aptamer to determine ligand concentration.

In one aspect, the disclosure provides a method of regulating geneexpression, the method comprising providing a ligand of an aptamer toany aptamer, DNA sequence, aptamer-regulated device, vector, or celldescribed herein, under conditions where an actuator acts on a geneticsequence of interest to regulate gene expression. In some embodiments,the genetic sequence of interest encodes a reporter protein. In someembodiments, the genetic sequence of interest encodes a therapeuticprotein. In some embodiments, the genetic sequence of interest is anRNA-based therapeutic. In some embodiments, the cell is a mammaliancell, bacterial cell, fungal cell, algal cell, or plant cell.

In one aspect, the disclosure provides a ligand of folinic acid, afolate, or a derivative thereof for use in regulating gene expression.In one aspect, the disclosure provides a ligand of folinic acid, afolate, or a derivative thereof for use in modulating RNA expression. Inone aspect, the disclosure provides a ligand of folinic acid, a folate,or a derivative thereof for use in modulating protein expression. In oneaspect, the disclosure provides an aptamer, DNA sequence,aptamer-regulated device, vector, or cell for use in regulating geneexpression. In one aspect, the disclosure provides an aptamer, DNAsequence, aptamer-regulated device, vector, or cell for use inmodulating RNA expression. In one aspect, the disclosure provides anaptamer, DNA sequence, aptamer-regulated device, vector, or cell for usein modulating protein expression.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. The patent orapplication file contains at least one drawing executed in color. Copiesof this patent or patent application publication with color drawing(s)will be provided by the Office upon request and payment of the necessaryfee. It is emphasized that, according to common practice, the variousfeatures of the drawings are not to-scale. On the contrary, thedimensions of the various features are arbitrarily expanded or reducedfor clarity. Included in the drawings are the following figures.

FIG. 1. Workflow diagram for de novo, in vitro selection of RNA aptamersensors and their integration into synthetic RNA regulatory switches.Design considerations, control parameters, and validation measurementsassist in achieving process milestones and increase the likelihood forsuccessful aptamer and switch function, particularly in intracellularenvironments.

FIG. 2. Folinic acid as a clinical drug. Chemical structures of(6S)-Folinic Acid and (6R)-Folinic Acid.

FIG. 3. Controlling selection stringency during de novo, in vitroselection of folinic acid-binding RNA aptamers. Binding enrichment ofRNA library to folinic acid-derivatized column over course of selectionis observed through monitoring fraction of ³²P-radiolabeled RNA librarythat binds to column and is specifically eluted off column.Concentration of folinic acid conjugated onto column is decreased by10-fold every other round to increase selection stringency.

FIG. 4A-4H. Predicted secondary structures of truncated aptamersequences characterized and corresponding surface plasmon resonancesensorgrams for (6R)-folinic acid and (6S)-folinic acid binding. (FIG.4A-4E) (6R)-folinic acid-specific aptamers FA8-4 (SEQ ID NO:18), FA8-11(SEQ ID NO:20), FA8-3 (SEQ ID NO:19), FA6-8 (SEQ ID NO:21), and FA8-16(SEQ ID NO:22), respectively. (FIG. 4F-4H) (6S)-folinic acid-specificaptamers FA8-18 (SEQ ID NO:23), FA10-7 (SEQ ID NO:24), and FA8-14 (SEQID NO:7), respectively.

FIG. 5A-5C. Screening of a transmitter library isolates switches thatregulate gene expression in response to folinic acid.

FIG. 6A-6C. Folinic acid-responsive switches isolated from transmitterscreen regulate gene expression in vivo. (FIG. 6A, 6B) Stranddisplacement mechanism for (6R)-FA-switch1 (SEQ ID NO:88) and(6R)-FA-switch2 (SEQ ID NO:89), respectively. Active ribozymeconformation with unbound folinic acid aptamer is shown on left, andinactive ribozyme conformation with bound folinic acid aptamer is shownon right. Nucleotides that comprise the sensor, actuator, andtransmitter components are colored blue, black, and orange,respectively. Folinic acid is represented by red polygon. Shadedpositions within transmitter component are randomized to generate devicelibraries. (FIG. 6C) Gene regulatory activity in S. cerevisiae. Switchesmaintain aptamer-conferred specificity for (6R)-folinic acid over(6S)-folinic acid in vivo.

FIG. 7. Synthesis for coupling small molecule amines to folinic acid.

FIG. 8A-8C. Validation of small molecule amine coupling to folinic acid.Folinic acid was covalently coupled to n-butylamine in the presence ofEDC, NHS, and MES buffer. (FIG. 8A) In the absence of the coupling agentEDC, LC-MS analysis detected only unreacted folinic acid. (FIG. 8B) Inthe presence of EDC, LC-MS analysis detected both single and doubleaddition product formation and complete conversion of folinic acidsubstrate into amine-coupled products. (FIG. 8C) Integrated peak valuesfor chromatograms shown in panels (FIG. 8A) and (FIG. 8B). n.d.=notdetected.

FIG. 9. Synthesis for coupling folinic acid to amino-modified solidsupport beads.

FIG. 10A-10C. Quantifying ligand concentration on solid support beadsafter coupling reaction. (FIG. 10A) Representative folinic acid standardcurve measured at 274 nm. (FIG. 10B) LC chromatogram for conjugationquantification. ˜95% conjugation efficiency seen for coupling 1M folinicacid and 0.1M folinic acid. (FIG. 10C) Integrated peak values forchromatograms shown in panel (FIG. 10B). Concentration of folinic acidremaining in reaction solution is calculated using linear regression fitline of standard curve prepared and run along with reaction samples.Percentage of folinic acid reacted is calculated by subtractingconcentration remaining after 2 hrs from initial concentration added.Concentration of folinic acid conjugated onto solid support beads iscalculated as twice the difference between the initial concentrationadded and concentration remaining in reaction solution after 2 hrs, asvolume of beads is one-half total reaction volume.

FIG. 11. Selection column RNA retention assay. RNA library was runthrough derivatized and non-derivatized amino-functionalized SepharoseEAH beads to determine optimal immobilization procedure to minimizenonspecific binding between RNA and beads. Unmodified beads showed highretention of RNA (˜82% of input RNA) after washing with 7 column volumesof selection buffer, while blocking beads with acetate significantlyreduced nonspecific binding of RNA onto column to 1.46%. Similarly,incomplete derivitization of amino groups on beads with folinic acid(either 0.2 mM FA or 2 mM FA), yielded retention of ˜⅔ of input RNA ontocolumn after washing (manufacturer states 7-12 mM amino groups onbeads). However, performing an additional acetate blocking reactiondirectly after folinic acid immobilization reaction decreased RNAretention to 1.3-1.4%. Nonspecific binding between RNA library and beadsis due to electrostatic attraction between positively charged aminogroup at selection buffer pH 7.4 and negatively charged phosphatenucleic acid backbone.

FIG. 12A-12C. In vitro selection enriches for high affinity sequencesthat bind folinic acid. Approximate dissociation constant (K_(D)) valuesare for (6R,S)-folinic acid.

FIG. 13A-13H. Truncation of folinic acid aptamers from full-lengthsequences isolated from in vitro selection. Predicted secondarystructures of full-length sequences are shown, with nucleotides incharacterized truncated sequences shown in blue. Of particular interestare truncated sequences for aptamers FA8-18 and FA10-7, which fold intopredicted secondary structures not present in lowest energy predictedsecondary structure of full-length sequence. (FIG. 13A-13E) (6R)-folinicacid-specific aptamers FA8-4 (SEQ ID NO:2), FA8-11 (SEQ ID NO:4), FA8-3(SEQ ID NO:3), FA6-8 (SEQ ID NO:5), and FA8-16 (SEQ ID NO:6),respectively. (FIG. 13F-13H) (6S)-folinic acid-specific aptamers FA8-18(SEQ ID NO:7), FA10-7 (SEQ ID No:8), and FA8-14 (SEQ ID NO:9),respectively. Circled nucleotide in FA8-4 in panel (FIG. 13A) waschanged to a U to facilitate base pairing in characterized truncatedsequences without affecting binding affinity.

FIG. 14A-14B. Truncation of aptamer FA8-14 (SEQ ID NO:25). Trucatedaptamer FAt8-14 retains ability to bind folinic acid but its irregularsensorgram curvature, seen in its trends of not reaching a steadyequilibrium plateau and exhibiting negative response values duringdissociation phase, preclude proper fitting to a 1:1 binding model anddetermination of kinetic and equilibrium binding properties.

FIG. 15. Sequence alignment of folinic acid aptamers. Randomized regionsof full-length sequences (omitting constant primer-binding regions) werealigned using ClustalX. Aptamers FA8-4, FA8-16, FA8-11, and FA6-8 sharea consensus sequence motif, while other aptamers do not share homologywith one another. Sequences from Top to Bottom: SEQ ID NOs:90-97.

FIG. 16. Comparison of aptamer kinetic properties. In vitro selectedaptamers for folinic acid exhibit k_(on) rates among the fastest ofcharacterized aptamers and k_(off) rates slower than most in vitroselected aptamers characterized and within range observed for naturalaptamers. In vitro selected and natural aptamer data reproduced fromreference (2) and include natural aptamers for glycine, c-di-GMP classI, c-di-GMP class II, TPP Thi1, and TPP thiM and in vitro selectedaptamers for theophylline, malachite green, flavin mononucleotide,arginine, citrulline, tyrosine, and ATP. Range of kinetic values forarginine, citrulline, tyrosine, and ATP aptamers are estimated based oninstrument specifications and measured equilibrium dissociationconstants. Representative aptamer sensorgrams shown, clockwise frombottom left: glycine, TPP Thi1, folinic acid FA8-18, theophylline, andATP. All kinetic binding values were determined under identicalconditions: 10 mM HEPES, 150 mM NaCl, 5 mM MgCl₂, pH 7.4, 25° C.

FIG. 17A-17H. Sequence-activity relationship study of folinic acidaptamer FAt8-4. Stem sequence constraints and variable positions werestudied to identify minimal binding sequences and aid facile integrationof aptamers into regulatory switch platforms. Single-stranded, unlabeledcircles represent variable nucleotide positions. Base-paired, unlabeledcircles represent nucleotide positions that were variable from parentaptamer based on nucleotide substitutions mentioned in materials andmethods section and are possibly either variable as long as base pairingis maintained or are optional base pairs. FAt8-4-stem1: SEQ ID NO:98;Fat8-4-stem2: SEQ ID NO:99; FAt8-4-stem3: SEQ ID NO:100; andFAt8-4stem4: SEQ ID NO:101.

FIG. 18. Sequence-activity relationship studies of folinic acidaptamers. Stem sequence constraints and variable positions were studiedto identify minimal binding sequences and aid facile integration ofaptamers into regulatory switch platforms. Single-stranded, unlabeledcircles represent variable nucleotide positions. Base-paired, unlabeledcircles represent nucleotide positions that were variable from parentaptamer based on nucleotide substitutions mentioned in materials andmethods section and are possibly either variable as long as base pairingis maintained or are optional base pairs. Nucleotides conserved betweenaptamers FA8-4 and FA8-11 are indicated in orange. Black lines indicateoptional base pairs. FA8-4: SEQ ID NO:102; FA8-11: SEQ ID NO:103; FA8-3:SEQ ID NO:104; and FA10-7: SEQ ID NO:105

FIG. 19. Structure-activity relationship studies of folinic acid ligandbinding. Binding contributions of the formyl and glutamate moieties offolinic acid were studied using folate derivatives. Replacement of theformyl functional group with a methyl group resulting in decreasedaptamer affinity, suggesting that the formyl oxygen is involved inhydrogen bonding contacts with one or more nucleotides of the aptamer.Elimination of the glutamate moiety of folinic acid did not impairbinding, providing support that the glutamate residue does notsignificantly participate in binding interactions.

FIG. 20. Strand displacement mechanism for rationally designed folinicacid-responsive switches. FA8-3-switch9: SEQ ID NO:106; andFA8-4-switch4:SEQ ID NO:107.

FIG. 21. Rationally designed folinic acid-responsive, ribozyme-baseddevices regulate gene expression in vivo in yeast.

FIG. 22. Strategy for generating ribozyme-based switches throughreplacement of ribozyme loop with aptamer. Ribozyme is integratedthrough helix III into 3′ UTR of regulated transcript of gene ofinterest (G01) (shown in gray). Device library is generated by replacingeither loop I or loop II with an aptamer (shown in orange) andrandomizing nucleotides of other loop (shown in green). Functionalswitches are isolated that reconstitute the tertiary ribozyme loop-loopinteraction as a tertiary aptamer-loop interaction (shown in blue),enabling ribozyme cleavage in the absence of aptamer ligand. In thepresence of ligand, aptamer-ligand binding competitively sequestersnucleotides involved in aptamer-loop interaction, disrupting tertiaryinteraction, preventing ribozyme cleavage, and allowing translation andexpression of gene.

FIG. 23. Device library for folinic acid aptamer loop replacementstrategy.sTRSV:SEQ ID NO:108; 6bpL1N7: SEQ ID NO:109; 6bpL1N6: SEQ IDNO:110; 6bpL1 FA8-4: SEQ ID NO:111; 6bpL1FA8-11: SEQ ID NO:112; 3bpL1N7:SEQ ID NO:113; 5bpL1FA8-4: SEQ ID NO:114; 5bpL1FA8-11: SEQ ID NO:123;4bpL2FA8-4: SEQ ID NO:115; 4bpL2FA8-11: SEQ ID NO:116; 4bpL2N6: SEQ IDNO:117; 3bpL2N7: SEQ ID NO:118; 3bpL2N6: SEQ ID NO:119; 5bpL2FA8-4: SEQID NO:120; 4bpL2FA8-11: SEQ ID NO:121; and 3bpL2N7: SEQ ID NO:122.

FIG. 24A-24B. Loop replacement screen for folinic acid switches.

FIG. 25A-25G. Folinic acid-responsive switches isolated from loopreplacement screen.

FIG. 25A: SEQ ID NO:124; FIG. 25B: SEQ ID NO:125; FIG. 25E: SEQ IDNO:126; and FIG. 25F: SEQ ID NO:127.

FIG. 26A-26C. Folinic acid-responsive miRNAs. (FIG. 26A) Predictedsecondary structure of folinic acid-responsive miRNA switch. Aptamernucleotides are highlighted in red. Blue arrows indicate sites of Droshaenzymatic cleavage. SEQ ID NO:128. (FIG. 26B) Dose-dependent silencingactivity of a single copy of the miRNA switch. (FIG. 26C) Silencingactivity of two copies of the miRNA switch.

DETAILED DESCRIPTION OF THE INVENTION

Engineered biological systems hold promise in addressing pressing humanneeds in chemical processing, energy production, materials construction,and maintenance and enhancement of human health and the environment.However, significant advancements in the ability to engineer biologicalsystems have been limited by the foundational tools available forreporting on, responding to, and controlling intracellular components inliving systems. In particular, sensors that respond to clinically usefulligands in vivo are needed.

The present invention provides an aptamer-regulated framework forengineering ligand-controlled gene-regulatory systems. An aptamer of theinvention is provided as a stand-alone entity, in a device coupled to anactuator, or in a device coupled to a transmitter component and anactuator, which actuator includes without limitation ribozymes, miRNA,etc. A variety of modes of standardized information transmission betweenthe ligand, aptamer and actuator can be employed. For example, theseswitch platforms may be applied to the construction of transgenicregulatory control systems that are responsive to folinic acid, afolate, or derivatives thereof. In regulating sets of functionalproteins, these switches can act to rewire information flow throughcellular networks and reprogram cellular behavior in response to changesin the ligand concentration. In regulating reporter proteins, theaptamer-regulated devices can serve as synthetic cellular sensors tomonitor temporal and spatial fluctuations in ligand levels. Due to theirgeneral applicability, these platforms offer broad utility forapplications in synthetic biology, biotechnology, and health andmedicine.

The limited set of available aptamers with validated function in vivo isdominated by only a few ligands and is an acute bottleneck in preventingthe wider application of RNA-based control devices. In contrast arenumerous approaches for constructing synthetic regulatory switches fromexisting aptamers, including rational design, in vitro selection,genetic screens and selections, fluorescence-activated cell sorting(FACS), cell motility, and in silico computational approaches, and thediversity of regulatory mechanisms available such as transcription,splicing, RNA stability, RNA interference, translation, andpost-translational activity.

De novo generation of RNA aptamer sensors through in vitro selectionprocesses is a particularly powerful approach for constructing novelinducible gene expression systems but has remained a considerablechallenge. Likely reasons so few in vitro selected aptamers havevalidated function in regulatory switches in vivo are that in vitroselected components often exhibit reduced activity when transferred toan intracellular environment and that aptamers must be amenable tophysical integration into a regulatory platform, without compromisingtheir binding activity. In vivo usage presents additional challenges asit requires high aptamer affinity and specificity, low ligand toxicity,sufficient ligand solubility and membrane permeability for exogenouslysupplied ligands, minimal ligand metabolism or degradation, lack ofinterference from intracellular molecules and RNAs, and functionalaptamer binding at physiological conditions (e.g., temperature, pH, andsalt concentrations, particularly Mg²⁺ levels). These difficulties haveresulted in only a handful of in vitro selected aptamers beingconsistently used and successfully incorporated into cellulargene-regulatory devices, even with the abundance of regulatory switchplatforms developed.

Riboswitches and devices are described in, for example, U.S. Pat. Nos.8,603,996; 8,604,176; and 8,772,464, each herein specificallyincorporated by reference.

Definitions

Folinic acid, folates, and derivatives thereof. The aptamers of thepresent invention are responsive to folinic acid and related compoundsas ligands. Shown in FIG. 2, folinic acid is chemically synthesized as amixture of two diastereomeric isomers, (6S)- and (6R)-folinic acid andis approved for clinical use either as the diastereomeric mixtureleuocovorin or as the single (6S) diastereomer levoleucovorin. The(6R)-folinic acid is heterologous and not biologically active, it ismetabolized slower, extending its plasma half-life and concentration upto 15-fold and 18-fold, respectively, compared to the (6S) diastereomer.The low toxicity and biological stability of (6R)-folinic acid make it aparticularly suitable ligand for RNA-based gene regulation.

Folates include, without limitation, derivatives with altered specificfunctionalities of folinic acid, which is composed of pterin,para-aminobenzoate, glutamate, and formyl moieties (FIG. 19). Forexample, (6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid replaces the5-formyl group with a 5-methyl group, testing the role of the formyloxygen on binding. (6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acidremoves the glutamate group through hydrolysis of the amide bond. Theglutamate group can be altered or removed without a loss of affinity tothe aptamer, thus in some embodiments of the invention(6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acid is useful as a ligand.Folates also include changes in oxidation states, for example folicacid, dihydrofolic acid. The aptamers of the invention also find use inindirect screening for antifolate drugs, e.g., methotrexate, pemetrexed,etc. Folates include, but are not limited to, folic acid, dihydrofolicacid, 5-formiminotetrahydrofolate, 10-formyltetrahydrofolate,5,10-methenyltetrahydrofolate, 5,10-methylenetetrahydrofolate,levomefolic acid, technetium (99mTc) etarfolatide, tetrahydrofolic acid,antifolates, vintafolide, methotrexate, pemetrexed, tetrahydrofolicacid, (6R)-tetrahydrobiopterin, (6R,S)-5-methyl-5,6,7,8-tetrahydrofolicacid, (6R)-5-methyl-5,6,7,8-tetrahydrofolic acid,(6S)-5-methyl-5,6,7,8-tetrahydrofolic acid,(6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acid,(6R)-5-formyl-5,6,7,8-tetrahydropteroic acid, and(6S)-5-formyl-5,6,7,8-tetrahydropteroic acid.

For screening and monitoring purposes, the chemical functional groupspresent within folinic acid are amenable for covalent coupling, e.g. tocolumns, reporter molecules, radioactive labels, and the like. Since theglutamate moiety of folinic acid is not necessary for ligand binding,tagged derivatives of folinic acid can be synthesized, using theglutamate residue of folinic acid or the benzoic acid of5-formyl-5,6,7,8-tetrahydropteoric acid as a chemical handle to couple acargo such as a fluorophore tag. The availability of diverse folateanalogues can also be mined to test the effect of different oxidationstates of the carbon atoms in the 5, 6, 7, or 8 positions or ligandconformation through derivatives that bridge the 5′ and 10′ nitrogens.

Ligand: “Ligand” or “analyte” or grammatical equivalents herein is meantto refer to any folinic acid, folate, or derivative thereof, includingtagged derivatives, to be detected and that can interact with an aptamerof the invention.

The terms “nucleic acid molecule” and “polynucleotide” refer todeoxyribonucleotides or ribonucleotides and polymers thereof in eithersingle- or double-stranded form. The term “RNA” (such as the RNAcomprising one or more aptamers) refers to ribonucleic acid, preferablyin single-stranded form. Unless specifically limited, the termsencompass nucleic acids/RNAs containing known analogues of naturalnucleotides which have similar binding properties as the referencenucleic acid and are metabolized in a manner similar to naturallyoccurring nucleotides. The terms may also encompass nucleic acids/RNAscontaining chemical modifications, such as modifications at the basemoiety, sugar moiety, and/or phosphate backbone, that tend to increasestability or half-life of the molecules in vivo. For example, thesemolecules may have naturally occurring phosphodiester linkages, as wellas those having non-naturally occurring linkages, e.g., forstabilization purposes, or for enhancing hydrophobic interaction withprotein ligands. Polynucleotides may have any three dimensionalstructure, and may perform any function, known or unknown. The followingare non-limiting examples of polynucleotides: coding or non-codingregions of a gene or gene fragment, loci (locus) defined from linkageanalysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA),ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA(shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinantpolynucleotides, branched polynucleotides, plasmids, expression vectors,isolated DNA of any sequence, isolated RNA of any sequence, nucleic acidprobes, and primers. A polynucleotide may comprise one or more modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure may be impartedbefore or after assembly of the polymer. The sequence of nucleotides maybe interrupted by non-nucleotide components. A polynucleotide may befurther modified after polymerization, such as by conjugation with adetectable label.

Aptamer. As used herein, the terms “aptamer(s)” or “aptamer sequence(s)”are meant to refer to single stranded nucleic acids (RNA or DNA) whosedistinct nucleotide sequence determines the folding of the molecule intoa unique three dimensional structure, allowing it to bind to a ligand ofthe invention at a high affinity. Aptamers comprising 15 to 120nucleotides can be selected from a pool of oligonucleotides. It will beunderstood by one of skill in the art that an aptamer sequence can beencoded and expressed from an expression construct, using vectors andpromoters known in the art.

Aptamers have specific binding regions which are capable of binding theligand in an environment wherein other substances in the sameenvironment are not bound to the nucleic acid. The specificity of thebinding is defined in terms of the comparative dissociation constants(K_(d)) of the aptamer for its ligand as compared to the dissociationconstant of the aptamer for other materials in the environment orunrelated molecules in general. A ligand is one which binds to theaptamer with greater affinity than to unrelated material. Typically, theK_(d) for the aptamer with respect to its ligand will be at least about10-fold less than the K_(d) for the aptamer with unrelated material oraccompanying material in the environment. Even more preferably, theK_(d) will be at least about 50-fold less, more preferably at leastabout 100-fold less, and most preferably at least about 200-fold less.An aptamer will typically be between about 10 and about 100 nucleotidesin length. More commonly, an aptamer will be between about 15 and about50 nucleotides in length, and truncated aptamer binding moietiesdisclosed herein are typically from about 15-75 nucleotides in length,and may be about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,or 85 nt. in length.

Although not necessary, an aptamer may include at least one modifiedbase moiety which is selected from the group including but not limitedto 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxytriethyl)uracil, 5-carboxymethylam inomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil; beta-D-mannosylqueosine,5-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Exemplary modified sugar moiety may be selectedfrom the group including but not limited to arabinose,2-fluoroarabinose, xylulose, and hexose. Exemplary modified ribose sugarmoiety may be selected from the group including but not limited to2′-fluoro, 2′-O-methyl, and 2′-O-alkyl. Exemplary neutral peptide-likebackbone modification include: peptide nucleic acid (PNA) (see, e.g., inPerry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670 and inEglom et al. (1993) Nature 365:566), locked nucleic acid (LNA), bridgednucleic acid (BNA), or modified phosphate backbone selected from thegroup consisting of a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

The aptamer of the invention can be comprised entirely of RNA. In otherembodiments of the invention, however, the aptamer can instead becomprised entirely of DNA, or partially of DNA, or partially of othernucleotide analogs. Such aptamer RNAs may be introduced into a cell as aDNA that encodes the aptamer such that transcription results in theaptamer-regulated RNA. Alternatively, an aptamer-regulated RNA itselfcan be introduced into a cell.

High affinity aptamers useful in the invention include those set forthin FIG. 13 as full—length or, preferably, truncated version. Predictedsecondary structures of full-length sequences are shown in FIG. 13, withnucleotides in characterized truncated sequences shown in blue.(6R)-folinic acid-specific aptamers are shown as FA8-4, FA8-11, FA8-3,FA6-8, and FA8-16 (FIGS. 13 a, b, c, d, e). (6S)-folinic acid-specificaptamers are shown as FA8-18, FA10-7, and FA8-14 (FIGS. 13 f, g, h). Thefull length sequences obtained from the in vitro selection process areshown in Table 4, and in FIG. 15 (which figure does not show primerbinding regions in the sequences).

Aptamers of the invention can comprise a degenerate sequence, and canfurther comprise fixed sequences flanking the degenerate sequence.Within the truncated sequence, certain residues are required forbinding, while others can be mutated, as shown in FIG. 18. For example,the circled nucleotides in the stem of FAt8-4-stem2 in FIG. 17b weremutated from the original FAt8-4 sequence, demonstrating that the entirebase stem can be mutated and still maintain binding activity. The basestem is still required to base pair, but the length of the stem (numberof base pairs) can be shortened as long as it still forms. In thesequences provided below, a dash intends that the intervening sequencecan be any sequence, or that a functional aptamer is formed by havingtwo separate strands hybridize together.

Exemplary is the truncated form of FA8-4, where the truncated form canbe designated FAt8-4. The original FA8-4 sequence is (shown in FIG. 13a):

(SEQ ID NO: 2) GGGACUUCUGCCCGCCUCCUUCCUGCUCGUGUCAAAAUGAAUGGCGCUCGGCGUUGCGUGGUACGUUAUAUUCCGGCCAAGCAGCCAUUCAUGGGAGACG AGAUAGGCGGACAC

From this initial sequence, the truncated form FAt8-4 is obtained (shownunderlined). The C shown in bold was also mutated to a U in FAt8-4, togive the truncated sequence:

(SEQ ID NO: 18) GCUUGGCGUUGCGUGGUACGUUAUAUUCCGGCCAAGC

Through stem mutations, it is shown that the nucleotides that providebinding activity are: GUUGCGUGGUACGUUAUAUUCCG. The minimally requiredsequence for binding is:

(SEQ ID NO: 129) N₁N₂GN₃UGCGUGGUACGUUAUAUUCCGN₄N₅,

where N₁ and N₅ are any complementary pair of nucleotides; N₂ and N₄ areany complementary pair of nucleotides; and N₃ is any nucleotide.

For FAt8-11, the minimal required sequence is:

(SEQ ID NO: 130) N₁N₂UGCN₃UGGUACGUUAUAUUCRGN₄N₅,

where N₁ and N₅ are any complementary pair of nucleotides; N₂ and N₄ areany complementary pair of nucleotides; N₃ is any nucleotide; and R is anA or G nucleotide.

For FAt8-3, the minimal required sequence is:

(SEQ ID NO: 131) CGUCUGGUCACGACCN₁N₂-N₃N₄CCCUCGAAAUCACGAGGGRG ACRAGAY,

where N₁ and N₄ are any complementary pair of nucleotides; N₂ and N₃ areany complementary pair of nucleotides; R is an A or G nucleotide; Y is aC or U nucleotide; and the dash indicates any intervening sequence ofnucleotides or two separate nucleotide strands.

For FAt10-7, the minimal required sequence is:

(SEQ ID NO: 132) N₁N₂GGCGAAGAGUCAAAGCAUCCCCN₃-N₄GGGCCCN₅N₆,where N₁ and N₆ are any complementary pair of nucleotides; N₂ and N₅ areany complementary pair of nucleotides; N₃ and N₄ are any complementarypair of nucleotides; and the dash indicates any intervening sequence ofnucleotides or two separate nucleotide strands.

Aptamers of the invention may be “substantially homologous” (or“substantially similar”) to the provided sequences, particularly withrespect to the truncated sequences provided in FIG. 13 and FIG. 14, andthe required residues shown in FIG. 18. Substantially similar variantsmay vary by a nucleotide substitution or deletion from a providedsequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 nucleotides. Such variants can have at least about 75%sequence similarity, at least about 80% sequence similarity, at leastabout 85% sequence similarity, at least about 90% sequence similarity,at least about 95% sequence similarity, at least about 99% sequencesimilarity, or more.

Aptamer sequences can be provided as an isolated nucleic acid, e.g. as asingle stranded RNA; can be provided as the encoding DNA sequence, whichmay be single stranded or double stranded; or may be operably joined toone or both of an actuator component and a transmitter component.

Actuator. An actuator is a component of aptamer-regulated devices of theinvention that provides for an activity, e.g. splicing activity;ribozyme activity; transcription; RNA stability; RNA interference;translation; post-translational activity; transcription termination; RNApolymerase recruitment; splice site accessibility; position-dependentsplicing; branch point sequence accessibility; ribozyme self-cleavage;Rnt1p enzymatic processing; Drosha pri-miRNA processing;ribozyme-mediated pri-miRNA folding; Dicer shRNA processing; ribosomalbinding site (RBS) accessibility; 16S ribosomal RNA; RBS accessibility;ribozyme-mediated RBS accessibility; ribozyme-mediated tRNA; tRNA; mRNA;ribosome binding, scanning, or assembly; antisense RNA binding to startcodon; TetR activity; promoter activity; terminator activity;riboswitches; ribozymes; microRNAs; siRNAs; antisense RNAs; RNAi;clustered regularly interspaced short palindromic repeats (CRISPR);CRISPR-Cas9; CRISPR targeting RNA (crRNA); trans-activating crRNA(tracrRNA); guide RNA (gRNA); splicing; small RNAs; ribosome bindingsites; internal ribosome entry sites; aptamer; etc. In some specificembodiments the actuator is a ribozyme, including without limitation ahammerhead ribozyme. In some embodiments, the actuator is an aptamer. Inother embodiments the actuator is an ampliswitch, as defined in U.S.Pat. No. 8,772,464, herein specifically incorporated by reference. Otheractuators of interest include ligand-responsive, microRNA (miRNA)switches that modulate Drosha processing. These switches integrate anaptamer into the basal segments of a miRNA. Internal loop size containedwithin the basal segments affects Drosha processing and therefore thelevels of miRNA-mediated gene silencing. By integrating an aptamerwithin the basal segments, unbound aptamer can remain relativelyunstructured, while ligand binding can sequester nucleotides involved inbinding and inhibit Drosha processing. Aptamer-regulated devicesinclude, but are not limited to, RNA switches, ribozyme switches,microRNA switches, an aptamer operably joined or linked to anotheraptamer, an aptamer operably joined or linked to an actuator, and anycombination thereof.

Ribozyme: Ribozymes are RNA molecules with a catalytic activity.Frequently, although not necessarily, the activity is cleavage of anucleic acid. Other ribozymes may catalyze other chemical reactions andhave their catalytic activity controlled using a similar type of switch.

Ribozyme types include, without limitation, hairpin ribozymes, hepatitisdelta virus and hepatitis delta virus-like ribozymes, CPEB3 ribozymes,Varkud satellite ribozymes, twister ribozymes, group I and group IIintrons, and hammerhead ribozymes. Ribozymes have been targeted to awide variety of substrates and tested in biological systems to achievethe inhibition of cellular gene expression or viral replication. Targetspecificity may be achieved, for example, by flanking a ribozyme motifwith antisense sequences, complementary to the target RNA.

A hammerhead ribozyme contains a core, three stems that extend from thecore, referred to herein as stem I, stem II, and stem III, and at leastone loop, which is located on the opposite end of a stem from the core.Hammerhead ribozymes can be type I, type II, or type III depending onwhich stem the ribozyme is integrated through into the transcript

As used herein, a “cis-cleaving hammerhead ribozyme” is a hammerheadribozyme that, prior to cleavage, is comprised of a singlepolynucleotide. A cis-cleaving hammerhead ribozyme is capable ofcleaving itself.

As used herein, a “trans-cleaving hammerhead ribozyme” is a hammerheadribozyme that, prior to cleavage, is comprised of at least twopolynucleotides. One of the polynucleotides is the target sequence thatis cleaved.

Ribozyme switches. A molecule that can adopt at least two differentconformational states, where each state is associated with a differentactivity of the molecule. Often a ligand can bind to one or moreconformations of the switch, such that the presence of the ligand shiftsthe equilibrium distribution across the adoptable conformations andtherefore regulates the activity of the switch molecule. As used here,switch generally refers to an RNA molecule that can adopt differentstructures that correspond to different gene-regulatory activities. AnRNA switch is then a ligand-controlled gene-regulatory system.

Switching Strand: The nucleic acid sequence within a strand displacementdomain that is bound to the general transmission region of the switchwhen the sensor domain is in the disrupted conformation (i.e., in theabsence of ligand). The switching strand is displaced by the competingstrand in the presence of ligand.

Strand displacement mechanism is demonstrated herein for architectureswith aptamer integration off of the hammerhead loop II, and integrationinto the transcript through hammerhead helix III (FIG. 20). Theseswitches demonstrate that selected folinic acid aptamers functionintracellularly and can be coupled to ribozymes to confer folinicacid-responsive gene regulation. For loop II integration, transmittersequences from previously characterized switches can be used as astarting point to join folinic acid aptamers to the hammerhead ribozyme,and modified as necessary to achieve proper folding of the ribozyme andaptamer domains. Exemplary switches are provided in Table 5.

Aptamer integration through helix III requires two integration stems onthe aptamer:

one for ribozyme helix III integration and another for transcriptintegration. In this architecture, the ribozyme sequence does not needto be altered, and therefore the native tertiary loop-loop interactionsare maintained, with the goal of achieving lower basal activity andeliminating the need to rescue impaired tertiary interactions.

An alternative switch architecture based on ribozyme loop replacementrelies on the observation that natural hammerhead ribozymes possess manysequence solutions for maintaining tertiary loop-loop interactions thatare crucial for stringent regulatory silencing (FIG. 22). Thisintegration strategy replaces one of the two interacting hammerheadloops with an aptamer, placing an internal or terminal loop of anaptamer approximately in the same position as the replaced ribozymeloop. To rescue the loop-loop interaction, the second loop is completelyrandomized and the device library is screened for functional switches.In the absence of ligand, nucleotides of the two loops are predicted tointeract through tertiary interactions. However, in the presence ofligand, ligand binding to the aptamer sequesters aptamer nucleotidesinvolved in the loop-loop interaction, precluding proper tertiarycontact formation and disrupting ribozyme cleavage.

The highest performing switches demonstrated up to 25.2-fold activationratios (ratio of GFP expression in the presence of ligand to expressionin the absence of ligand) in the presence of 3 mM (6R)-folinic acid anddynamic ranges of up to 43.6% (difference between GFP expression in thepresence and absence of ligand).

Stem: A stem is a nucleic acid motif that extends from the ribozymecore, at least a portion of which is double-stranded. In certainembodiments, there is a loop at the opposite end of the stem from theribozyme core, and this loop connects the two strands of thedouble-stranded stem. In certain embodiments, a stem comprises 2 to 20complementary base pairs. In certain embodiments, a stem comprises 3, 4,5, 6, 7, 8, or 9 complementary base pairs. Stems are numbered accordingto where they extend from the core sequence. In certain embodiments, ahammerhead ribozyme contains three stems, which are referred to as stemI stem II, and stem III.

In certain embodiments, at least 30% of the nucleotides in a stem arepart of a complementary base pair. The remaining base pairs may bemismatched, non-complementary base pairs, or may be part of a bulge. Incertain embodiments, at least 40% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 50% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, at least 60% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 70% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, at least 80% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 90% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, at least 95% of the nucleotides in a stem are partof a complementary base pair. In certain embodiments, at least 99% ofthe nucleotides in a stem are part of a complementary base pair. Incertain embodiments, 100% of the nucleotides in a stem are part of acomplementary base pair.

Loop: A loop is a sequence of nucleotides that is not paired withanother strand and is located at the distal end of a stem that isopposite the core. In certain embodiments, a loop is between 1 to 20nucleotides long. In certain embodiments, a loop is between 2 and 10nucleotides long. In certain embodiments, a loop is between 3 and 8nucleotides long. The loop is numbered according to the stem to which itis attached. Therefore, loop I is located at the end of stem I oppositethe core, loop II is located at the end of stem II opposite the core,and loop III is located at the end of stem III opposite the core.

As used herein, a “stem/loop” refers to the entire stem, along with anybulges within that stem, and the loop at the end of the stem. Forexample, stem/loop I includes stem I, including any bulges within stemI, and loop I. If a stem lacks a loop, then stem/loop refers to thestem, along with any bulges within that stem.

Bulge: As used herein, a “bulge” is a sequence of nucleotides that isnot paired with another strand and is flanked on both sides bydouble-stranded nucleic acid sequences. In certain embodiments, a bulgeis located within a stem. When a bulge is located within a stem, thenucleotides of the bulge are considered to be part of the stem. Incertain embodiments, a hammerhead ribozyme comprises more than onebulge. In certain embodiments, a bulge within a stem is located two basepairs from the core. In certain embodiments, one or both strands of thestem contain a bulge.

Directly Coupled: As used herein, an aptamer is directly coupled to anactuator, e.g., a loop of a ribozyme where the loop, relative to activeribozyme structure in the absence of the aptamer, is interrupted at onlyone backbone phosphodiester bond between two residues of the loop, thebackbone phosphodiester bond being replaced with phosphodiester bonds tothe 5′ and 3′ ends of the aptamer. In the active form of theaptamer-regulated ribozyme, the 5′ and 3′ residues of the informationtransmission domain are based paired to one another to form a duplexregion in order to preserve the structure of the otherwise interruptedloop.

Information Transmission Domain: A switch domain that encodes thefunction of transmitting information between the sensor domain and theactuator domain.

Information Transmission Mechanism: A general mechanism for transmittinginformation between the sensor domain and the actuator domain of aswitch. As used here, this mechanism regulates the activity of theactuator domain in response to the binding state of the sensor domain.

Strand Displacement Mechanism: An information transmission mechanismthat is based on the rational design of an information transmissiondomain that functions through a strand displacement event. Such a stranddisplacement event utilizes competitive binding of two nucleic acidsequences (the competing strand and the switching strand) to a generaltransmission region of the switch (the base stem of the aptamer) toresult in disruption or restoration of the actuator domain in responseto restoration of the sensor domain.

For convenient detection, aptamers, aptamer-regulated devices,polynucleotides, or compounds disclosed herein can be conjugated to adetectable label. Suitable detectable labels can include any compositiondetectable by photochemical, biochemical, spectroscopic, immunochemical,electrical, optical, or chemical means. A wide variety of appropriatedetectable labels are known in the art, which include fluorescentlabels, chemiluminescent labels, radioactive isotope labels, enzymaticlabels, and ligands. The detection methods used to detect or quantifythe label will typically depend upon the label selected. For example,radiolabels (e.g., radioactive isotope labels) may be detected usingPositron Emission Topography (PET), photographic film, or aphosphoimager. Fluorescent labels (e.g., fluorescent dyes, fluorescentproteins) may be detected and quantified using a photodetector to detectemitted light. In some embodiments, each of a plurality of aptamers,aptamer-regulated devices, polynucleotides, or compounds is conjugatedto a different detectable label (e.g., fluorescent dyes with differentemission spectra), such that the signal corresponding to differenttargets can be differentiated. Enzymatic labels are typically detectedby providing the enzyme with a substrate and measuring the reactionproduct produced by the action of the enzyme on the substrate.Colorimetric labels are typically detected by visualizing the coloredlabel or are quantified using a spectrophotometer.

In certain embodiments, the aptamers, aptamer-regulated devices,polynucleotides, or compounds disclosed herein are isotopically labeled.Isotopically-labeled aptamers, polynucleotides, or compounds (e.g., anisotopologue) may have one or more atoms replaced by an atom having adifferent atomic mass or mass number. Non-limiting examples of isotopesthat can be incorporated into the disclosed compounds include isotopesof hydrogen, carbon, nitrogen, oxygen, phosphorous, sulfur, fluorine,chlorine, and iodine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹³N, ¹⁵N, ¹⁵O, ¹⁷O,¹⁸O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, ¹²³I, and ¹²⁵I, respectively. Certainisotopically-labeled aptamers, polynucleotides, or compounds, forexample, those incorporating a radioactive isotope, are useful in drugand/or substrate tissue distribution studies. The radioactive isotopestritium (³H) and carbon-14 (¹⁴C) are particularly useful for thispurpose in view of their ease of incorporation and ready means ofdetection. These radiolabeled compounds could be useful to helpdetermine or measure the effectiveness of the compounds, bycharacterizing, for example, the site or mode of action, or bindingaffinity to a pharmacologically important site of action. Substitutionwith heavier isotopes such as deuterium (²H) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample, increased in vivo half-life or reduced dosage requirements, andhence are preferred in some circumstances. Substitution with positronemitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and ¹³N, can be useful inPositron Emission Topography (PET) studies for examining substratereceptor occupancy. Isotopically-labeled compounds can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein using an appropriateisotopically-labeled reagent in place of the non-labeled reagent.

In some embodiments, an aptamer, aptamer-regulated device, orpolynucleotide binds to a ligand with a K_(d) of about 100 pM, 200 pM,300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 2 nM, 3nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80nM, 85 nM, 90 nM, 95 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM,400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 950 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM,9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 150μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, or more than500 μM. In some embodiments, an aptamer, aptamer-regulated device, orpolynucleotide binds to a ligand with a K_(d) of up to about 100 pM, 200pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 2 nM,3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80nM, 85 nM, 90 nM, 95 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM,400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 950 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM,9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 150μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, or 500 μM. In someembodiments, an aptamer, aptamer-regulated device, or polynucleotidebinds to a ligand with a K_(d) of at least about 100 pM, 200 pM, 300 pM,400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 2 nM, 3 nM, 4 nM,5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30 nM, 35 nM,40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90nM, 95 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900nM, 950 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM,15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 150 μM, 200 μM,250 μM, 300 μM, 350 μM, 400 μM, 450 μM, or 500 μM. In some embodiments,an aptamer, aptamer-regulated device, or polynucleotide binds to aligand with a K_(d) within the range from 100 pM-100 μM, from 100 pM-10μM, from 100 pM-1 μM, from 100 pM-900 nM, from 100 pM-800 nM, from 100pM-700 nM, from 100 pM-600 nM, from 100 pM-500 nM, from 100 pM-400 nM,from 100 pM-300 nM, from 100 pM-200 nM, from 100 pM-100 nM, or from 100pM-50 nM. In some embodiments, an aptamer, aptamer-regulated device, orpolynucleotide binds to a ligand with a K_(d) of up to about 50 nM, 55nM, 60 nM, 65 nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 150nM, 200 nM, 250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, or 1 μM.

In some embodiments, an aptamer-regulated device achieves a half-maximalregulatory response at a concentration of a ligand of about 100 pM, 200pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 nM, 2 nM,3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15 nM, 20 nM, 25 nM, 30nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65 nM, 70 nM, 75 nM, 80nM, 85 nM, 90 nM, 95 nM, 100 nM, 150 nM, 200 nM, 250 nM, 300 nM, 350 nM,400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM,850 nM, 900 nM, 950 nM, 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM,9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, 100 μM, 150μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, or more than500 μM. In some embodiments, an aptamer-regulated device achieves ahalf-maximal regulatory response at a concentration of a ligand of up toabout 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM,900 pM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 15nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 150 nM, 200 nM,250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM,700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 μM, 2 μM, 3 μM, 4 μM,5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM,40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90μM, 95 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450μM, or 500 μM. In some embodiments, an aptamer-regulated device achievesa half-maximal regulatory response at a concentration of a ligand of atleast about 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800pM, 900 pM, 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM,15 nM, 20 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 55 nM, 60 nM, 65nM, 70 nM, 75 nM, 80 nM, 85 nM, 90 nM, 95 nM, 100 nM, 150 nM, 200 nM,250 nM, 300 nM, 350 nM, 400 nM, 450 nM, 500 nM, 550 nM, 600 nM, 650 nM,700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 μM, 2 μM, 3 μM, 4 μM,5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM, 15 μM, 20 μM, 25 μM, 30 μM, 35 μM,40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, 75 μM, 80 μM, 85 μM, 90μM, 95 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450μM, or 500 μM. In some cases, a regulatory response may include, but isnot limited to, RNA expression, protein expression, gene expression,fluorescence, binding, cell viability, cell proliferation, cellmotility, and any combination thereof.

In some embodiments, the ratio of a regulatory response in the presenceof a ligand to the regulatory response in the absence of the ligand ofan aptamer-regulated device is about 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6;1.7; 1.8; 1.9; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0;7.5; 8.0; 8.5; 9.0; 9.5; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 25;30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 200; 300;400; 500; 600; 700; 800; 900; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000;7,000; 8,000; 9,000; 10,000; or more than 10,000. In some embodiments,the ratio of a regulatory response in the presence of a ligand to theregulatory response in the absence of the ligand of an aptamer-regulateddevice is at least about 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8;1.9; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0;8.5; 9.0; 9.5; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 25; 30; 35;40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 200; 300; 400; 500;600; 700; 800; 900; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000;8,000; 9,000; 10,000; or more than 10,000. In some embodiments, theratio of a regulatory response in the presence of a ligand to theregulatory response in the absence of the ligand of an aptamer-regulateddevice is up to about 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9;2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5;9.0; 9.5; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 25; 30; 35; 40;45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 200; 300; 400; 500;600; 700; 800; 900; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000;8,000; 9,000; 10,000; or more than 10,000. In some cases, a regulatoryresponse is an activation ratio. In some cases, the aptamer-regulateddevice is modulating RNA expression, protein expression, geneexpression, or any combination thereof.

In some embodiments, the ratio of a regulatory response in the absenceof a ligand to the regulatory response in the presence of the ligand ofan aptamer-regulated device is about 1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6;1.7; 1.8; 1.9; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0;7.5; 8.0; 8.5; 9.0; 9.5; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19; 20; 25;30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 200; 300;400; 500; 600; 700; 800; 900; 1,000; 2,000; 3,000; 4,000; 5,000; 6,000;7,000; 8,000; 9,000; 10,000; or more than 10,000. In some embodiments,the ratio of a regulatory response in the absence of a ligand to theregulatory response in the presence of the ligand of anaptamer-regulated device is at least about 1.0; 1.1; 1.2; 1.3; 1.4; 1.5;1.6; 1.7; 1.8; 1.9; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5;7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19;20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100;200; 300; 400; 500; 600; 700; 800; 900; 1,000; 2,000; 3,000; 4,000;5,000; 6,000; 7,000; 8,000; 9,000; 10,000; or more than 10,000. In someembodiments, the ratio of a regulatory response in the absence of aligand to the regulatory response in the presence of the ligand of anaptamer-regulated device is up to about 1.0; 1.1; 1.2; 1.3; 1.4; 1.5;1.6; 1.7; 1.8; 1.9; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5; 5.0; 5.5; 6.0; 6.5;7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10; 11; 12; 13; 14; 15; 16; 17; 18; 19;20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80; 85; 90; 95; 100;200; 300; 400; 500; 600; 700; 800; 900; 1,000; 2,000; 3,000; 4,000;5,000; 6,000; 7,000; 8,000; 9,000; 10,000; or more than 10,000. In somecases, a regulatory response is an activation ratio. In some cases, theaptamer-regulated device is modulating RNA expression, proteinexpression, gene expression, or any combination thereof.

In some embodiments, an aptamer, aptamer-regulated device, orpolynucleotide binds to a ligand with a specificity of about 1.0; 1.1;1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.5; 3.0; 3.5; 4.0; 4.5;5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10; 11; 12; 13; 14;15; 16; 17; 18; 19; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70; 75; 80;85; 90; 95; 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000; 2,000;3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; 10,000; or more than10,000. In some embodiments, an aptamer, aptamer-regulated device, orpolynucleotide binds to a ligand with a specificity of at least about1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.5; 3.0; 3.5;4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10; 11; 12;13; 14; 15; 16; 17; 18; 19; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70;75; 80; 85; 90; 95; 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000;2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; 10,000; or morethan 10,000. In some embodiments, an aptamer, aptamer-regulated device,or polynucleotide binds to a ligand with a specificity of up to about1.0; 1.1; 1.2; 1.3; 1.4; 1.5; 1.6; 1.7; 1.8; 1.9; 2.0; 2.5; 3.0; 3.5;4.0; 4.5; 5.0; 5.5; 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0; 9.5; 10; 11; 12;13; 14; 15; 16; 17; 18; 19; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65; 70;75; 80; 85; 90; 95; 100; 200; 300; 400; 500; 600; 700; 800; 900; 1,000;2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; 10,000; or morethan 10,000. In some cases, specificity is calculated as (K_(d) of theaptamer to the ligand)/(K_(d) of the aptamer to a reference compound).In some cases, the reference compound is a compound that is not theligand and is selected from (6S)-folinic acid; (6R)-folinic acid; adiastereomeric mixture of (6R)- and (6S)-folinic acid; tetrahydrofolicacid; (6R)-tetrahydrobiopterin; folic acid; dihydrofolic acid;5-formiminotetrahydrofolate; 10-formyltetrahydrofolate;5,10-methenyltetrahydrofolate; 5,10-methylenetetrahydrofolate;levomefolic acid; technetium (99mTc) etarfolatide; tetrahydrofolic acid;vintafolide; methotrexate; pemetrexed; tetrahydrofolic acid;(6R)-tetrahydrobiopterin; (6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid;(6R)-5-methyl-5,6,7,8-tetrahydrofolic acid;(6S)-5-methyl-5,6,7,8-tetrahydrofolic acid;(6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acid;(6R)-5-formyl-5,6,7,8-tetrahydropteroic acid;(6S)-5-formyl-5,6,7,8-tetrahydropteroic acid; and any combinationthereof. In some cases, specificity is calculated as (K_(d) of theaptamer to (6R)-folinic acid)/(K_(d) of the aptamer to (6S)-folinicacid) or (K_(d) of the aptamer to (6S)-folinic acid)/(K_(d) of theaptamer to (6R)-folinic acid). In some cases, specificity is calculatedas (K_(d) of the aptamer to (6R)-folinic acid)/(K_(d) of the aptamer to(6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid) or (K_(d) of the aptamerto (6S)-folinic acid)/(K_(d) of the aptamer to(6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid). In some cases,specificity is calculated as (K_(d) of the aptamer to (6R)-folinicacid)/(K_(d) of the aptamer to (6R,S)-5-formyl-5,6,7,8-tetrahydropteroicacid) or (K_(d) of the aptamer to (6S)-folinic acid)/(K_(d) of theaptamer to (6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acid).

In some embodiments, an aptamer, aptamer-regulated device, orpolynucleotide has a length of about 1; 2; 3; 4; 5; 6; 7; 8; 9; 10; 11;12; 13; 14; 15; 16; 17; 18; 19; 20; 25; 30; 35; 40; 45; 50; 55; 60; 65;70; 75; 80; 85; 90; 95; 100; 200; 300; 400; 500; 600; 700; 800; 900;1,000; 2,000; 3,000; 4,000; 5,000; 6,000; 7,000; 8,000; 9,000; 10,000;or more than 10,000 nucleotides. In some embodiments, an aptamer,aptamer-regulated device, or polynucleotide has a length of up to 50;55; 60; 65; 70; 75; 80; 85; 90; 95; 100; 200; 300; 400; 500; 1,000; or2,000 nucleotides. In some embodiments, an aptamer has a length of up to50; 55; 60; 65; 70; 75; 80; 85; 90; 95; or 100 nucleotides.

In some embodiments, an aptamer, aptamer-regulated device, orpolynucleotide comprises a sequence selected from FAt8-4, FAt8-11,FAt8-3, FAt6-8, FAt8-16, FAt8-18, FAt10-7, and FAt8-14. In some cases,an aptamer, aptamer-regulated device, or polynucleotide comprises asequence having about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to a sequence selectedfrom FAt8-4, FAt8-11, FAt8-3, FAt6-8, FAt8-16, FAt8-18, FAt10-7, andFAt8-14. In some cases, an aptamer, aptamer-regulated device, orpolynucleotide comprises a sequence having at least about 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%identity to a sequence selected from FAt8-4, FAt8-11, FAt8-3, FAt6-8,FAt8-16, FAt8-18, FAt10-7, and FAt8-14. In some cases, an aptamer,aptamer-regulated device, or polynucleotide comprises a sequence havingup to about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or 100% identity to a sequence selected from FAt8-4,FAt8-11, FAt8-3, FAt6-8, FAt8-16, FAt8-18, FAt10-7, and FAt8-14.

In some cases, an aptamer, aptamer-regulated device, or polynucleotidecomprises a sequence having about 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity tosingle-stranded regions of a sequence selected from FAt8-4, FAt8-11,FAt8-3, FAt6-8, FAt8-16, FAt8-18, FAt10-7, and FAt8-14 as determined bythe secondary structure (e.g., as shown in FIG. 4, 13, 14, 17, or 18).

Delivery

RNA or nucleic acid molecules of the invention can be delivered totarget cells in vivo, through any convenient mechanism, includingwithout limitation transfection, infection, electroporation, etc. Targetcells may include plant cells, animal cells, prokaryotes, fungi, etc.Target cells may include bacteria, fungi, plant protoplasts andchloroplasts, and mammalian cell lines. Cells may include, but are notlimited, to S. cerevisiae, HeLa, CTLL-2, primary human T_(CM), HEK293,U2OS, SK-MEL-28, A549, Colo 829, NCH89, Vero, SK-MEL-28, Capan-1, A549,CHO, HepG2, X. laevis, A. baumannii, A. baylyi, A. tumefaciens, B.subtilis, E. coli, M. magneticum, M. smegmatis, S. pyogenes, Cucumberprotoplast, M. tuberculosis, M. smegmatis, S. coelicolor, S. elongatus,N. tabacum chloroplasts, Francisella. Vectors of interest include,without limitation, viral vectors, mini-circle vectors, plasmids andinclude episomal vectors that replicate autonomously, as well as vectorsthat are integrated into a chromosome of the cell. The initialintroduction of the nucleic acid can be performed in vitro, in vivo, orex vivo, as required for the specific use.

Vectors can be plasmid, viral, or others known in the art, used forreplication and expression in cells, e.g. mammalian cells, plant cells,etc. A promoter may be operably linked to the sequence encoding thesubject RNA. Expression of the subject encoded sequences can be by anypromoter known in the art to act in mammalian, preferably human cells.Such promoters can be inducible or constitutive. Such promoters includebut are not limited to: the SV40 early promoter region (Bernoist andChambon, Nature 290:304-310 (1981)), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell22:787-797 (1980)), the herpes thymidine kinase promoter (Wagner et al.,Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)), the regulatorysequences of the metallothionine gene (Brinster et al, Nature 296:3942(1982)), etc. Any type of plasmid, cosmid, YAC, mini-circle or viralvector can be used to prepare the recombinant DNA construct that can beintroduced directly into the tissue site. Alternatively, viral vectorscan be used which selectively infect the desired tissue, in which caseadministration may be accomplished by another route (e.g.,systematically).

Another approach utilizes a recombinant DNA construct in which the RNAor other aptamer-containing nucleic acid is placed under the control ofa strong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of the subject RNA. For example, a vector orexpression construct can be introduced in vivo such that it is taken upby a target cell and directs the transcription of a subject RNA. Such avector or expression construct can remain episomal or becomechromosomally integrated, as long as it can be transcribed to producethe desired product. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art.

Thus the invention also provides an expression vector or constructhaving a coding sequence that is transcribed to produce one or moretranscriptional products that produce a subject RNA in the treatedcells. Expression vectors appropriate for producing an aptamer-regulatednucleic acid are well-known in the art. For example, the expressionvector is selected from an episomal expression vector, an integrativeexpression vector, and a viral expression vector.

In certain embodiments, the expression vector can be designed to includeone or more subject RNA an RNA transcript, such as in the 3′untranslated region (3′-UTR), so as to regulate transcription, stabilityand/or translation of that RNA transcript in a manner dependent on theligand. To further illustrate, the expression construct can include acoding sequence for a polypeptide such that the mRNA transcript includesboth the polypeptide coding sequence as well as one or more of the RNAof the invention. In this way, expression of the polypeptide can berendered dependent on the ligand(s) to which the aptamer(s) bind.

An aptamer, aptamer-regulated device, or polynucleotide described hereincan be obtained using chemical synthesis, molecular cloning orrecombinant methods, DNA or gene assembly methods, artificial genesynthesis, PCR, or any combination thereof. Methods of chemicalpolynucleotide synthesis are well known in the art and need not bedescribed in detail herein. One of skill in the art can use thesequences provided herein and a commercial DNA synthesizer to produce adesired DNA sequence. For preparing polynucleotides using recombinantmethods, a polynucleotide comprising a desired sequence can be insertedinto a suitable cloning or expression vector, and the cloning orexpression vector in turn can be introduced into a suitable target cellfor replication and amplification, as further discussed herein.Polynucleotides may be inserted into target cells by any means known inthe art. Cells may be transformed by introducing an exogenouspolynucleotide, for example, by direct uptake, endocytosis,transfection, F-mating, chemical transformation, or electroporation.Once introduced, the exogenous polynucleotide can be maintained withinthe cell as a non-integrated expression vector (such as a plasmid) orintegrated into the target cell genome. The polynucleotide so amplifiedcan be isolated from the target cell by methods well known within theart. Alternatively, PCR allows reproduction of DNA sequences.

RNA can be obtained by using the isolated DNA in an appropriateexpression vector and inserting it into a suitable target cell. When thecell replicates and the DNA is transcribed into RNA, the RNA can then beisolated using methods well known to those of skill in the art.Alternatively, RNA can be obtained by transcribing the isolated DNA, forexample, by an in vitro transcription reaction using an RNA polymerase.Alternatively, RNA can be obtained using chemical synthesis.

Suitable cloning vectors may be constructed according to standardtechniques, or may be selected from a large number of cloning vectorsavailable in the art. While the cloning vector selected may varyaccording to the target cell intended to be used, useful cloning vectorswill generally have the ability to self-replicate, may possess a singletarget for a particular restriction endonuclease, and/or may carry genesfor a marker that can be used in selecting clones containing theexpression vector. Suitable examples include plasmids and bacterialviruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and itsderivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNAs, andshuttle vectors such as pSA3 and pAT28. These and many other cloningvectors are available from commercial vendors such as BioRad,Strategene, and Invitrogen.

In one aspect, the disclosure provides an expression vector comprisingany of the aptamers, aptamer-regulated devices, or polynucleotidesdisclosed herein described herein. An aptamer, aptamer-regulated device,or polynucleotide disclosed herein may be located in an expressionvector. An expression vector may be a construct, which is capable ofdelivering, and preferably expressing, one or more gene(s) orsequence(s) of interest in a target cell. Examples of expression vectorsinclude, but are not limited to, viral vectors (e.g., adenoviruses,adeno-associated viruses, and retroviruses), naked DNA or RNA expressionvectors, plasmids, cosmids, phage vectors, DNA or RNA expression vectorsassociated with cationic condensing agents, DNA or RNA expressionvectors encapsulated in liposomes, and certain eukaryotic cells, such asproducer cells. An expression vector may allow easy and efficientreplication, cloning, and/or selection. Accordingly, an expressionvector may additionally include nucleic acid sequences that permit it toreplicate in the target cell, such as an origin of replication, one ormore therapeutic genes and/or selectable marker genes and other geneticelements known in the art such as regulatory elements directingtranscription, translation and/or secretion of the encoded protein.Expression vector components may generally include, but are not limitedto, one or more of the following: a signal sequence; an origin ofreplication; one or more marker genes; and suitable transcriptionalcontrolling elements (such as promoters, enhancers and terminator). Forexpression (e.g., translation), one or more translational controllingelements are also usually required, such as ribosome binding sites,translation initiation sites, internal ribosome entry site, and stopcodons. The expression vector may be used to transduce, transform orinfect a cell, thereby causing the cell to express nucleic acids and/orproteins other than those native to the cell. The expression vectoroptionally includes materials to aid in achieving entry of the nucleicacid into the cell, such as a viral particle, liposome, protein coatingor the like. Numerous types of appropriate expression vectors are knownin the art for protein expression, by standard molecular biologytechniques. Such expression vectors are selected from among conventionalvector types including insects, e.g., baculovirus expression, or yeast,fungal, bacterial or viral expression systems. Other appropriateexpression vectors, of which numerous types are known in the art, canalso be used for this purpose. Methods for obtaining cloning andexpression vectors are well-known (see, e.g. Green and Sambrook,Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring HarborLaboratory Press, New York (2012)).

In one aspect, the disclosure provides a target cell comprising any ofthe aptamers, aptamer-regulated devices, or polynucleotides describedherein. In one aspect, the disclosure provides a target cell comprisingany of the expression vectors described herein. Any target cell capableof over-expressing heterologous DNA can be used. Suitable target cellsinclude, but are not limited to, mammalian (e.g., human such as HEK,HEK293, HEK293T, HeLa, U205, SK-MEL-28, A549, Colo 829, NCH89, Hep G2,Capan-1, T cell, B cell, or primary human T_(CM); mouse such as a 3T3,CTLL-2, or cells derived from Swiss, BALB/c or NIH mice; hamster such asCHO; monkey such as COS or Vero), bacterial (e.g., Escherichia coli,Bacillus subtilis, Pseudomonas, Streptomyces, A. baumannii, A. baylyi,A. tumefaciens, M. magneticum, M. smegmatis, S. pyogenes, M.tuberculosis, M. smegmatis, S. coelicolor, Francisella), algae,cyanobacteria (e.g., S. elongatus), fungal (e.g., Saccharomycescerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis), amphibian(e.g., Xenopus laevis), plant (e.g., Arabidopsis, Cucumber protoplast,N. tabacum chloroplasts), or insect (e.g., Drosophila melanogaster, HighFive, Spodoptera frugipedera Sf9) target cells. A target cell mayinclude, but is not limited to, a cell from a mammal, human, non-humanmammal, domesticated animal (e.g., laboratory animals, household pets,or livestock), non-domesticated animal (e.g., wildlife), dog, cat,rodent, mouse, hamster, cow, bird, chicken, fish, pig, horse, goat,sheep, rabbit, and any combination thereof. In some cases, a target cellis from a human.

The target cells can be transfected, e.g. by conventional means such aselectroporation with at least one expression vector of the disclosure.The expression vectors containing the polynucleotides of interest can beintroduced into a target cell by any of a number of appropriate means,including electroporation, chemical transformation, transfectionemploying calcium chloride, rubidium chloride, calcium phosphate,DEAE-dextran, or other substances; microprojectile bombardment;lipofection; and infection (e.g., where the vector is an infectiousagent such as vaccinia virus). The choice of introducing vectors orpolynucleotides will often depend on features of the target cell. Thetransfected or transformed target cell may then be cultured underconditions that allow expression of the polynucleotide.

Formulations

The subject RNA or nucleic acids may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,polymers, receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.The subject RNA or nucleic acids can be provided in formulations alsoincluding penetration enhancers, carrier compounds and/or transfectionagents.

Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations which canbe adapted for delivery of the subject RNA or nucleic acid moleculesinclude, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844;5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020;5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804;5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978;5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152;5,556,948; 5,580,575; and 5,595,756.

The subject RNA or nucleic acids may also encompass any pharmaceuticallyacceptable salts, esters or salts of such esters, or any other compoundwhich, upon administration to an animal including a human, is capable ofproviding (directly or indirectly) the biologically active metabolite orresidue thereof. Accordingly, for example, the disclosure is also drawnto RNAs or nucleic acids and pharmaceutically acceptable salts, andother bioequivalents.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium potassium, magnesium,calcium, and the like. Examples of suitable amines areN,NI-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66,1-19). The base addition salts of said acidic compoundsare prepared by contacting the free acid form with a sufficient amountof the desired base to produce the salt in the conventional manner. Thefree acid form may be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention.

As used herein, a “pharmaceutical addition salt” includes apharmaceutically acceptable salt of an acid form of one of thecomponents of the compositions of the invention. These include organicor inorganic acid salts of the amines. Preferred acid salts are thehydrochlorides, acetates, salicylates, nitrates and phosphates. Othersuitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids. Preferred examples of pharmaceutically acceptable saltsinclude but are not limited to (a) salts formed with cations such assodium, potassium, lithium, ammonium, magnesium, calcium, polyaminessuch as spermine and spermidine, etc.; (b) acid addition salts formedwith inorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) saltsformed from elemental anions such as chlorine, bromine, and iodine.

Uses

The subject RNA or nucleic acids can further be used to engineer novelregulatory pathways and control loops for applications in metabolicengineering and synthetic circuit design by enabling a cell to sense andrespond to folinic acid levels. Because the activity of the subject RNAor nucleic acids may be tunable over a range of ligand concentrations,the system can be designed to inhibit or activate genes only whencertain triggering ligands have exceeded or gone below certain thresholdconcentrations. Balancing heterologous gene expression in biosyntheticpathways to maximize product yield can be achieved with the subject RNAor nucleic acids that regulate expression of any gene of interest inresponse to any pre-determined pathway intermediates, including anydisease markers. Synthetic gene circuits have recently been used tounderstand and model cellular networks and to achieve cellular controlas a step towards “programmable” cell behavior. Gene circuits can bebuilt using combinations of the subject RNA or nucleic acids asregulators for precise control schemes. The subject RNA or nucleic acidsare useful in building and characterizing circuits.

Finally, aptamer-regulated nucleic acids present tools for cellularimaging (by, for example, using a fluorescent reporter gene as the geneof interest), measuring, and detection strategies enabling programmableconcentration-specific detection of ligands. The subject RNA or nucleicacids offer a unique platform to generate tailor-made cellular sensorsand “smart” regulators that can target any gene in response to theligand, creating new avenues for cellular control and engineering.

EXPERIMENTAL Example 1 Programmable and Conditional Gene Control by aClinical Drug

Natural biological systems rely on the ability to sense and respond tomolecular and environmental stimuli through dynamic regulation of geneexpression. Engineering synthetic biological systems similarly requirescomparable sensing and control capabilities, particularly for effectorligands orthogonal to those found in nature or suitable for specializedapplications such as clinical therapeutics. The construction ofsynthetic RNA regulatory switches responsive to the clinical drugfolinic acid (leucovorin) is described herein. RNA aptamer sensors thatbind with high specificity and affinity to either the (6R)- or(6S)-diastereomers of folinic acid were generated de novo through invitro selection. Selection design principles were applied to favordiscovery of high affinity aptamers that function under intracellularconditions, requiring as few as 21 nucleotides and with dissociationconstants as low as 19.2 nM and ligand specificities up to 70,000-foldover structurally similar molecules. To engineer synthetic RNA switchesthat induce gene expression in vivo, a library of hammerheadribozyme-based switches based on a transmitter strand displacementmechanism was designed and screened for functional gene regulatoryactivity in yeast. The successful construction of folinicacid-responsive switches demonstrates the in vivo application of theseaptamers, which increases the number of orthogonal input signalsavailable for inducible gene expression and provides a much neededclinically relevant ligand for advancing cellular and RNA-basedtherapeutics. This system combines design considerations, controlparameters, and validation measurements to demonstrate a streamlinedprocess for constructing RNA switches to desired molecular targets thatcan extend their use in varied synthetic biology applications.

A general and integrated process for generating inducible, RNA-basedgene expression systems is described herein. A series of designprinciples, control parameters, and validation measurements weredeveloped for the de novo, in vitro selection of RNA aptamer sensorsthat increase the likelihood of enriching and isolating bindingsequences that function in vivo (FIG. 1). This system was applied toaddress a specific deficiency in available effector ligands, the lack ofclinically relevant inducers, by selecting a set of aptamers that bindthe clinical drug folinic acid. A recently reported surface plasmonresonance platform was used to validate and characterize aptamer bindingand to rapidly prototype aptamer integration sites (Chang et al. (2014)Analytical Chemistry 86(7):3273-3278). Using a FACS-based screeningmethod, a high affinity folinic acid aptamer was integrated into ahammerhead ribozyme through a randomized transmitter sequence andisolate functional switches that activate gene expression upon ligandinduction (Liang et al. (2012) Nucleic Acids Research 40(20):e154). Thiswork systematizes overall design principles for generating newbiological sensing capabilities that function in vivo and provides anintegrated process for generating and incorporating these sensingfunctions into genetically encoded, ligand-responsive, andgene-regulatory elements. These improvements in selection design andswitch integration provide a more robust strategy for generating geneticswitches to desired chemical targets for customizable inducible geneexpression.

Materials and Methods

Selection column preparation. Positive selection columns functionalizedwith folinic acid and negative selection columns without folinic acidwere prepared using EAH Sepharose 4B beads (GE Healthcare, Uppsala,Sweden). 0.5 mL beads per selection column were rinsed sequentially withwater (pH 4.5) and 500 mM NaCl. For conjugation reaction, (6R,S)-Folinicacid (Sigma-Aldrich, St. Louis, Mo.) was coupled through its carboxylategroups to amino-functionalized beads using an amide bond-formingcarbodiimide coupling reaction (FIG. 9). Rinsed beads were washed withconjugation buffer (0.1M MES, pH 6.0). N-hydroxysuccinimide (NHS, ThermoScientific) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC, Sigma-Aldrich) were dissolved in 0.5 mL conjugationbuffer to final concentrations of 0.06 M and 0.1 M, respectively,supplemented with appropriate folinic acid concentration between 1 μMand 1 mM depending on selection round (FIG. 2), and added to rinsedbeads for a total reaction volume of 1 mL per selection column. Reactionwas mixed thoroughly and incubated at room temperature for 2 hours withrotation. Remaining amine functional groups on beads were subsequentlyreacted with acetate to block nonspecific electrostatic interactionsbetween positively charged amine groups and negatively charged RNAbackbone. Likewise, negative selection beads without folinic acidconjugated were also blocked with acetate for negative selections. Forblocking reaction, folinic acid-conjugated beads or rinsed beads forpositive and negative selection columns, respectively, were washed withblocking buffer (1 M acetate, pH 6.0) and reacted with NHS and EDC asdescribed above but in the absence of folinic acid and with thereplacement of conjugation buffer with blocking buffer. All aqueoussolutions were prepared using water treated with 0.1% (v/v) diethylpyrocarbonate (Sigma-Aldrich) to inactivate RNases. 0.5 mL of positiveor negative selection beads were packed into 2 mL disposable polystyrenecolumns (Thermo Scientific) according to manufacturer's instructions,equilibrated with selection buffer (10 mM HEPES, 150 mM NaCl, pH 7.4, GEHealthcare, supplemented with 5 mM MgCl₂, Life Technologies, Carlsbad,Calif.), and stored at 4° C. until used.

Validation and quantification of coupling reaction. Folinic acidconjugation was validated and quantified by liquid chromatography massspectrometry (LC-MS). For conjugation validation, 5 mM folinic acid wascoupled to 5 mM n-butylamine (Sigma-Aldrich) in conjugation buffer with0.02 M NHS and 0.1 M EDC for 2 hours at room temperature, and 5 μL ofthe reaction mixture (diluted 50-fold) was separated on a Zorbax SB-Aqcolumn (3.0×50 mm, 1.8 μM particle size) (Agilent Technologies). Forconjugation quantification, beads from conjugation reactions werepelleted by centrifugation, and 5 μL of the reaction supernatant(diluted 10-fold for 1 mM folinic acid reactions) was separated on theZorbax column. The column was equilibrated with water, 0.1% acetic acid,and 0.1% methanol (Solvent A), and samples were eluted with a mobilephase of methanol and 0.1% acetic acid (Solvent B) in the followingsequence: 0-1 min. at 100% A, 1-4 min. 0-25% B, 4-7 min. at 25% B,followed by steps to clean the column with 100% B and re-equilibrate inA. The flow rate was held constant at 0.6 mL/min. Eluted reactionproducts were identified by UV absorbance at 274 nm or on an Agilent6320 Ion Trap mass spectrometer. For validation, extracted ionchromatograms of major reaction products were consistent with masses ofexpected coupling products (FIG. 7, FIG. 8). For quantification, peakarea of the UV or extracted ion chromatograms was integrated andcompared to standard curve of folinic acid.

De novo, in vitro aptamer selection. Selection library N70 antisensetemplate DNA oligonucleotide5′-GTGTCCGCCTATCTCGTCTCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGAAGGAGGCGGGCAGAAGTCCCTATAGTGAGTCGTATTAGAA (SEQ ID NO:1) was synthesized by Integrated DNATechnologies. Initial RNA library for first selection round covered˜1.2*10¹⁵ unique sequences and was transcribed by annealing 2 pmol DNAtemplate with 2.5 μmol primer N70-T7-fwd(5′-TTCTAATACGACTCACTATAGGGACTTCTGCCCGCCTCCTTCC) (SEQ ID NO:30) to forma partially double stranded template for run-off transcription using aMEGAshortscript T7 transcription kit (Life Technologies), supplementedwith 0.5 μCi α-³²P-GTP (MP Biomedicals, Solon, Ohio), according tomanufacturer's instructions. RNA libraries for subsequent selectionrounds used double stranded DNA template generated from PCRamplification. Unincorporated nucleotides were removed using NucAwaySpin Columns (Life Technologies), according to manufacturer'sinstructions. Purified RNA was resuspended in selection buffer,denatured at 65° C. for 5 min, and cooled to room temperature directlybefore use.

Selection columns were equilibrated at room temperature with 5 mLselection buffer, supplemented with 10 μg/mL yeast tRNA (Ambion) tosaturate nonspecific RNA binding sites. For selection rounds 1 and 2,RNA library was added to the positive selection column, which was thenwashed with appropriate volume of selection buffer supplemented with 10μg/mL yeast tRNA (FIG. 2). For selection rounds 3-10, a negativeselection column was inserted directly above the positive selectioncolumn to remove RNA sequences that bound to the beads. RNA library wasadded to the negative selection column, and the negative selectioncolumn was washed with 4 column volumes of selection buffer supplementedwith 10 μg/mL yeast tRNA before being removed and remaining bufferwashes (FIG. 2) applied to positive selection column.

Positive selection column was washed with 6 column volumes of selectionbuffer supplemented with appropriate folinic acid concentration (FIG. 2)to competitively elute bound RNAs off of column. Each wash and elutionvolume was collected separately, and the radiation intensity for eachcollected volume and selection column was measured using a ratemeter(Ludlum Measurements, Sweetwater, Tex.).

Elution volumes were pooled together, and RNA was concentrated usingAmicon Ultra-0.5 Centrifugal Filter Units with Ultracel-10 membranes(EMD Millipore, Billerica, MA). Concentrated RNA was reverse transcribedusing Superscript III Reverse Transcriptase (Life Technologies) withprimer N70-rev according to manufacturer's instructions, and cDNAproduct was PCR amplified using primers N70-fwd-short and N70-rev.Unincorporated nucleotides were removed using NucAway Spin Columns.Purified PCR product was used as template for RNA transcription reactionfor next selection round.

Individual sequences from selection rounds 6, 8, and 10 were isolated byPCR amplifying selection round libraries with primersGAP-N70-Avr11-fwd-short and GAP-N70-XhoI-rev and cloning into plasmidpCS1748 (1) via homologous recombination gap-repair transformation intoyeast using standard lithium acetate heat shock to generate singlecolonies. Individual yeast colonies were incubated at 99° C. for 10 minin 20 mM NaOH to lyse cells, and 5 μL of mixture were used at templatein PCR with primers CS653 and CS654. PCR product was purified usingEconoSpin columns (Epoch) and sequenced using primer CS653 (Elim).

Full length sequences were amplified off of sequencing PCR productsusing primers N70-fwd-short and Biacore-N70-rev to append a poly(A)tail. Truncated or mutated sequences were synthesized by Integrated DNATechnologies or the Protein and Nucleic Acid facility (Stanford,Calif.).

Surface plasmon resonance characterization of aptamer bindingproperties. Nucleic acid aptamer preparation. RNA aptamers were preparedby PCR amplification of DNA template sequences (Table 4, Integrated DNATechnologies, Coralville, Iowa) using forward and reverse primersBiacore-fwd and Biacore-rev, respectively, followed by transcription ofthe PCR product using the MEGAshortscript T7 transcription kit (LifeTechnologies) and purification of the transcription product using theRNA Clean & Concentrator kit (Zymo Research, Irvine, Calif.), accordingto the manufacturers' instructions. Aptamers were resuspended inselection buffer, denatured at 65° C. for 5 min, and cooled to roomtemperature directly before use.

Sensor chip surface generation. Experiments were performed on a BiacoreX100 instrument (GE Healthcare) at 25° C. A CM5 sensor chip (GEHealthcare) was equilibrated with HBS-N buffer. The DNA linker strand(5′-AmMC6-TTTTTTTTTTTTTTTTTTTTTTTT) (Integrated DNA Technologies) (SEQID NO:133), with an amino modified 6-carbon linker on the 5′ end, wasimmobilized to the chip surface. The carboxymethylated dextran surfaceof the CM5 chip was activated for 7 min at a flow rate of 10 μL/minusing a 1:1 volume ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (GE Healthcare) and 0.1 M N-hydroxysuccinimide (GEHealthcare). A molar ratio of 1:30 of DNA strand tohexadecyltrimethylammonium bromide (Sigma-Aldrich, St. Louis, Mo.) wasdiluted in 10 mM HEPES buffer (Sigma-Aldrich) to a final concentrationof 20 μM and 0.6 mM, respectively, and injected over the activatedsurface for 10 min at a flow rate of 5 μL/min. Excess activated groupswere blocked by an injection of 1 M ethanolamine (GE Healthcare), pH8.5, for 7 min at a flow rate of 10 μL/min. The immobilization reactionwas performed sequentially on both flow cells (FC1, FC2) and yieldedapproximately 4,000 RU of the DNA strand.

Aptamer binding assay. The Biacore X100 instrument was primed threetimes with running buffer prior to all binding assays. For each assay,three startup cycles were performed to stabilize the sensorgrambaseline. For each startup cycle, the aptamer (˜40-70 ng/μL, ˜3 μM) wascaptured onto the sample flow cell (FC2) for 40 s at a flow rate of 5μL/min. 25 mM NaOH (GE Healthcare) was injected for 30 s at a flow rateof 30 μL/min over both flow cells to regenerate the sensor surface. Adilution series of (6R)- or (6S)-folinic acid (Schircks Laboratories,Jona, Switzerland) was prepared in selection buffer and filtered througha 0.2 μm membrane (Pall Corporation, Port Washington, N.Y.). For eachconcentration sample, the aptamer was captured onto the sample flow cell(FC2) for 40 s at a flow rate of 5 μL/min, the folinic acid solution wasinjected over both flow cells at a flow rate of 30 μL/min to monitortarget association, and selection buffer was injected over both flowcells at a flow rate of 30 μL/min to monitor target dissociation.Association and dissociation phase lengths used were chosen based ontime needed to reach equilibrium. Aptamer and folinic acid were removedfrom the sensor surface by injecting 25 mM NaOH for 30 s at a flow rateof 30 μL/min over both flow cells.

Data processing and analysis were performed using Biacore X100Evaluation Software version 2.0 (GE Healthcare). A double-referencingmethod was performed to process all datasets. Data from the sample flowcell (FC2) were referenced first by subtracting data from the referenceflow cell (FC1) to correct for bulk refractive index changes,nonspecific binding, injection noise, matrix effects, and baselinedrift. Reference-subtracted data (FC2-FC1) were double-referenced with ablank injection of running buffer to account for any systematic driftover the course of the injection. Double-referenced data were fit to a1:1 binding model for kinetic analysis or steady-state affinity modelfor thermodynamic analysis. Reported values are the mean and standarddeviation of at least three independent experiments.

Plasmid and strain construction. Standard molecular biology cloningtechniques were used to construct all plasmids. DNA synthesis wasperformed by Integrated DNA Technologies (Coralville, Iowa) or theProtein and Nucleic Acid Facility (Stanford, Calif.). All enzymes,including restriction enzymes and ligases, were obtained through NewEngland Biolabs (Ipswich, Mass.). Ligation products were electroporatedwith a GenePulser XCell (Bio-Rad, Hercules, Calif.) into an E. coliDH10B strain (Invitrogen, Carlsbad, Calif.), where cells harboringcloned plasmids were maintained in Luria-Bertani media containing 50μg/mL ampicillin (EMD Chemicals, Philadelphia, Pa.). All clonedconstructs were sequence verified by Elim Biopharmaceuticals (Hayward,Calif.).

The two-color screening plasmid pCS1748; single-color plasmids harboringGFP (pCS1585) and mCherry (pCS1749) for use as compensation controls;and no-color plasmid pCS4, containing no fluorescence reporter gene, wasused as the negative-control construct. Ribozyme-based devices andappropriate controls were inserted into the 3′ untranslated region (UTR)of yEGFP3 in pCS1748 through appropriate restriction endonuclease andligation-mediated cloning. DNA fragments encoding the ribozyme-baseddevices and controls were PCR amplified using forward and reverseprimers L1-2-fwd and L1-2-rev, respectively, and inserted into pCS1748via the unique restriction sites AvrII and XhoI, which are located 3 ntsdownstream of the yEGFP3 stop codon. Cloned plasmids were transformedinto the budding yeast Saccharomyces cerevisiae strain W303α (MAT αhis3-11, 15 trp1-1 leu2-3 ura3-1 ade2-1) through a standard lithiumacetate method. All yeast strains harboring cloned plasmids weremaintained on synthetic complete media with a uracil dropout solutioncontaining 2% dextrose (SC-URA) and grown at 30° C. All plasmidsconstructed in this study are summarized in Table 3.

Cell sorting screen for functional ligand-responsive RNA switches.Device libraries (FIG. 17) were amplified using forward and reverseprimers GAP-L1-2-fwd and GAP-L1-2-rev, respectively. The library wasinserted into pCS1748 through homologous recombination-mediatedgap-repair during transformation into yeast strain W303. Briefly, an 800μL library PCR reaction was performed with 160 pmol of each primer and16 pmol of the library template. 8 μg of the plasmid pCS1748 wasdigested with AvrII and XhoI. The digested vector was combined with thelibrary PCR product, extracted with phenol chloroform, and precipitatedinto a dry pellet with ethanol. A Tris-DTT buffer (2.5 M DTT, 1 M Tris,pH 8.0) was added to a 50 mL yeast culture (OD₆₀₀ 1.3-1.5) and incubatedat 30° C. for 10-15 min. The yeast were pelleted, washed with chilledBuffer E (10 mM Tris, pH 7.5, 2 mM MgCl₂, 270 mM sucrose), andresuspended in Buffer E to a final volume of 300 μL. The yeast mixturewas directly added to the precipitated DNA pellet and 50 ρL of themixture was transferred to a chilled 2 mm gap cuvette forelectroporation (540 V, 25 μf, 1000 0). Following transformation, thecells were resuspended in 1 mL warmed YPD media and incubated at 30° C.for 1 hr. Multiple transformations (˜5) were performed to cover thedesired library diversity (˜10⁶-10⁷). Transformation efficiencies weredetermined by plating serial dilutions of the transformants, andtransformed cells were propagated in SC-URA media for FACS.

Two-color FACS-based screen. Cells harboring the RNA device librariesand control constructs were washed, resuspended in FACS buffer (1% BSAin PBS), and stained with a DAPI viability dye (Invitrogen). The cellsuspension was filtered through a 40 pm cell strainer (BD Systems, SanJose, Calif.) prior to analysis on a FACSAria II cell sorter (BDSystems). GFP was excited at 488 nm and measured with a splitter of 505nm and bandpass filter of 525/50 nm. mCherry was excited at 532 nm andmeasured with a splitter of 600 nm and a bandpass filter of 610/20 nm.DAPI was excited at 355 nm and measured with a bandpass filter of 450/50nm. A scatter gate was set based on the forward and side-scatter area ofcells harboring the negative-control plasmid (pCS4) to exclude debris,followed by a DAPI-(−) viability gate to exclude dead cells in theDAPI-(+) gate from the analysis. Cells harboring the single-colorcontrol plasmids (pCS1585, pCS1749) were analyzed to compensatespillover from GFP to the mCherry detector. A general sorting strategywas followed in which cells harboring devices with targetedgene-regulatory activities were analyzed to set a sorting gate on atwo-dimensional scatter plot that correlates GFP and mCherryfluorescence. Cells within this gate were collected into SC-URA mediaand propagated to sufficient density for further screening or analysis.

Device characterization through flow cytometry analysis. Enriched devicelibraries from FACS were grown overnight in SC-URA liquid media and thenplated onto SC-URA solid medium. Individual colonies were screened andcharacterized for gene-regulatory activity of the devices based on flowcytometry analysis. The GFP fluorescence was measured on a MACSQuant VYBflow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). DAPI wasexcited at 405 nm and measured with a filter of 450/50 nm. GFP wasexcited at 488 nm and measured with a filter of 525/50 nm. mCherry wasexcited at 561 nm and measured with a filter of 615/20 nm. Cellsharboring the mCherry-only plasmid (pCS1749) were analyzed to measurecellular autofluorescence in the GFP channel. Gene-regulatory activitieswere reported as relative GFP expression levels and were determined asthe median of the GFP fluorescence using FlowJo (Tree Star), andnormalized to the median of the GFP fluorescence of a positive control(sTRSV Contl, a noncleaving sTRSV ribozyme with a scrambled core) thatis grown under identical ligand conditions, run in the same experiment,and set to 100%.

Devices that exhibited desired activities were amplified by colony PCRusing forward and reverse primers CS653(5′-GGTCACAAATTGGAATACAACTATAACTCT) (SEQ ID NO:50) and CS654(5′-CGGAATTAACCCTCACTAAAGGG) (SEQ ID NO:51), respectively, andsequenced. The recovered devices were resynthesized and recloned intothe vector backbone to confirm the observed activity. DNA oligos weresynthesized and amplified for insertion into pCS1748 using forward andreverse primers L1-2-fwd and L1-2-rev, respectively. The resynthesizeddevices were inserted into pCS1748 via the unique restriction sitesAvrII and XhoI. The reconstructed device plasmids were transformed intothe W303 yeast strain through a standard lithium acetate method. Cellsharboring the selected devices and appropriate controls were prepared asdescribed above for the sorting experiments and analyzed to characterizethe gene-regulatory activity of each device. FlowJo was used to processall flow cytometry data. Gene-regulatory activities in the absence orpresence of ligand were determined as the median of the GFP fluorescencebased, and normalized to the median of the GFP fluorescence of apositive control (sTRSV Contl, a noncleaving sTRSV ribozyme with ascrambled core) in the absence or presence of ligand, respectively, tocorrect for any nonspecific effects of ligand on the measuredfluorescence (FIG. 19). Reported gene-regulatory activities are the meanand standard deviation of at least three independent experiments.

Results and Discussion

De novo, in vitro selection enriches RNA aptamer sensors to folinicacid. A distinct limitation in the implementation of synthetic RNAregulatory switches is the lack of clinically relevant small moleculeligands that are nontoxic and readily bioavailable and display favorablepharmacokinetic properties. The lack of clinically suitable effectorsrestricts the development of genetically controlled cellulartherapeutics and limits potential animal or human studies. For instance,theophylline-responsive switches have been implemented inproof-of-concept studies for regulating T cell proliferation foradoptive transfer therapy and viral replication and infectivity for genetherapy, cancer treatment, or vaccine development. However, its toxicityand narrow therapeutic index make theophylline unsuitable for clinicaluse.

One promising candidate for clinical use is the folate derivativefolinic acid (FIG. 2), which is used in cancer treatment in combinationwith the antimetabolite chemotherapy drugs methotrexate and5-fluorouracil and is naturally occurring. While only its (6S) form isbiologically active, folinic acid is chemically synthesized as a mixtureof two diastereomeric isomers, (6S)- and (6R)-folinic acid, and isapproved for clinical use either as the diastereomeric mixtureleuocovorin or as the single (6S) diastereomer levoleucovorin. As(6R)-folinic acid is heterologous and not biologically active, it ismetabolized slower, extending its plasma half-life and concentration upto 15-fold and 18-fold, respectively, compared to the (6S) diastereomer.The low toxicity and biological stability of (6R)-folinic acid make it aclinically suitable orthogonal input signal for RNA-based therapies. Inaddition, the chemical functional groups present within folinic acid areamenable for covalent coupling to column-based chromatographicseparation methods commonly used for in vitro selection (FIG. 2).

A continuous N70 randomized region was chosen as the basis for theselection library to provide sufficient sequence diversity and length asmany previously validated in vitro selected, small molecule bindingaptamers range in length from ˜30-60 nucleotides. Previous studies havesuggested N70 as being a particularly favorable length that increasesthe frequency that a given motif could occur while avoiding potentialinhibitory effects of excess sequence that could interfere with properfolding of a functional sequence, and more compact sequences arepreferred for engineering synthetic switches. Constant regions flankingthe randomized region were included as primer binding sites for RNAtranscription, reverse transcription, and PCR amplification andreproduced from a previous study (29). The initial RNA library pool of1.2×10¹⁵ unique sequences was generated through run-off transcription toprevent PCR amplification bias.

With the desired goal of functioning intracellularly, selectionconditions were chosen to mimic physiological environments, with pH of7.4 and moderate divalent magnesium concentration of 5 mM that wouldpromote RNA folding and binding. The selection buffer was limited toonly four components, Na⁺, Mg²⁺, Cl⁻, and HEPES, to minimize bindingdependence on the presence of a particular buffer component and tomaintain consistent selection and characterization conditions.

A recent aptamer selection meta-analysis observed correlation betweenselection immobilization concentration used during in vitro selectionand resulting aptamer binding affinities, with aptamers withdissociation constants under 1 μM having immobilization concentrationsof ˜400-500 μM. A sub-micromolar binding affinity was targeted for invivo applications, with a preferred goal of under 100 nM. Most in vitroselections use a constant immobilization concentration throughout theselection process. High concentrations of immobilized ligand decreasethe chance that functional binders will be lost but generally yieldlower affinity aptamers; low concentrations of immobilized ligandincrease the chance that binding sequences will be lost before they canbe enriched but yield higher affinity aptamers. A high concentration ofimmobilized ligand (2 mM) was used at the start to provide moreavailable ligand for the initial RNA library to bind to and to preventbeing too stringent initially and losing rare sequences but to steadilydecrease immobilization concentration down to 2 μM to favor highaffinity binders over weakly binding sequences (FIG. 3, Table 3).Chemical coupling to an amino-functionalized Sepharose bead support inimmobilization reactions was validated and its yield quantified as ˜95%;importantly, this yield was consistently observed regardless of ligandconcentration, enabling the resulting concentration of immobilizedligand to be confidently adjusted based on reaction ligand concentration(FIG. 7, FIG. 8, FIG. 10, FIG. 9). Unreacted bead amine groups wereblocked with acetate to prevent nonspecific binding of RNA to the column(FIG. 11). Elution concentrations of free ligand were also decreasedthroughout the course of the selection to minimize any possible effectof high concentrations of ligand counter ion (Ca²⁺ for the folinic acidused) on aiding nonspecific RNA elution while remaining at least12.5-fold higher than immobilization concentrations to out-compete offbinders from column (FIG. 3, Table 3). A similar strategy could be usedto decrease Mg²⁺ dependence gradually throughout the selection but wasnot implemented in this case, as selection for high affinity has beenposited to subsequently result in decreased magnesium dependence.

Selection stringency was controlled through multiple parameters andsteadily increased throughout the selection rounds. 2 mM folinic acidwas conjugated onto the column beads, and the concentration ofimmobilized ligand was decreased 10-fold every other round, reaching 2μM folinic acid on the beads by round 7 and held constant through round10. Wash volume was increased progressively from 6 column volumes inround 1, by an additional 3 column volumes each round (FIG. 3, Table 3).High transcription yields encouraged competition among RNA sequences forfewer binding sites.

Binding enrichment, as measured by percentage of RNA eluted, generallyincreased among successive rounds when ligand immobilizationconcentration on beads was held constant and dropped when ligandconcentration decreased (FIG. 3, Table 3). Negative selections against anon-derivatized column were included starting in round 3 to preventenrichment of RNA binding to the Sepharose bead support, which is ageneral problem encountered for chromatography-based affinity methods(FIG. 3). By constantly applying negative selective pressure againstSepharose binding, the percentage of RNA remaining on the negativeselection column remained under 1% for the course of the selection(Table 3); in contrast, in the absence of negative selection, enrichmentof RNA binders to the bead support was observed, emphasizing theimportance of selecting against this alternative and undesired bindingsolution.

Aptamer screening and truncation using surface plasmon resonanceisolates unique functional sequences. After 10 rounds of in vitroselection, binding of the library pool to a column derivatized withfolinic acid was observed (FIG. 3, Table 3). Bulk binding of the librarypools from rounds 8, 9, and 10 was confirmed by using a label-free,surface plasmon resonance (SPR)-based aptamer characterization platform(FIG. 12). Individual library sequences were isolated and sequencedafter cloning into a sequencing plasmid in a yeast host by homologousrecombination. Individual sequences were then amplified from sequencingplasmids, transcribed into RNA, and screened by SPR for binding andspecificity. Aptamer sequences were truncated to identify minimalsequences necessary for ligand binding and predicted, functionalsecondary structures (FIG. 4, FIG. 13). Rational truncations based offof predicted secondary structures were tested by truncating sequenceswhere stable stems formed (e.g., aptamers FAt8-4, FAt8-11, and FAt8-3).Sequence alignment identified homology among aptamers FA8-4, FA8-11,FA6-8, and FA8-16 with the shared consensus motif5′-GG(U)ACGUUAU(A)UUCNG (SEQ ID NO:134), where N is any nucleotide andnucleotides in parentheses are optional, that aided in truncating theseaptamers (FIG. 15).

For aptamers where rational truncations did not produce functionalbinders, serial truncations were performed where 5 to 10 nucleotideswere systematically removed from either the 5′ or 3′ ends. However,systematic truncations often produced false negatives, where sequencesthat contained the final truncated aptamer, and therefore would beexpected to be capable of binding, displayed no binding, presumably dueto improper folding. Exploring suboptimal predicted secondary structuresrevealed alternative folding structures that were tested to revealfunctional aptamer conformations (FIG. 13). For instance, proper foldingof the minimal FAt10-7 aptamer is disfavored in the full-length sequenceby 2.3 kcal/mol and is the twelfth lowest energy predicted structure(−41.8 kcal/mol for the lowest energy predicted structure vs. −39.5kcal/mol for the functional conformation). Likewise, proper folding ofthe minimal FAt8-18 aptamer is disfavored in the full-length sequence by2.2 kcal/mol and is the twenty-fifth lowest energy predicted structure(−29.5 kcal/mol for the lowest energy predicted structure vs. −27.3kcal/mol for the functional conformation). These predicted energiessuggest that folinic acid binding may contribute at least 2.2 kcal/molto stabilize these suboptimal folding conformations. Of eight aptamerscharacterized, only one, FA8-14, resisted truncation attempts; while atruncated sequence FAt8-14 maintains binding, it displays aunconventional binding response that cannot be fit to a 1:1 bindingmodel (FIG. 14).

Characterization of aptamer kinetic and equilibrium binding propertiesobserves high aptamer affinity and specificity. Binding properties oftruncated aptamer sequences to both (6R)- and (6S)-folinic acid werecharacterized using a label-free, surface plasmon resonance-based method(FIG. 4, Table 1). Aptamers demonstrated high affinity and specificity,with dissociation constants (Kos) as low as 19.2 nM and greater than70,000-fold selectivity between the two folinic acid diastereomers. Allaptamers displayed selectivity of at least 260-fold, and aptamers withKos below 65 nM were identified for both diastereomers. No aptamers wereidentified with similar affinities for both folinic acid diastereomers,likely a consequence from selective pressure for high affinity binders.Importantly, the Kos exhibited are significantly below concentrations ofthe drug that can accumulate in human blood plasma through intravenousinfusion, up to 77.6 μM for (6R)-folinic acid and 7.95 μM for(6S)-folinic acid, indicating that these aptamers should be sufficientlysensitive to operate within the human body for cellular and therapeuticapplications.

Folinic acid aptamers exhibit k_(on) rates among the fastest ofcharacterized natural or in vitro selected aptamers, k_(off) ratesslower than most in vitro selected aptamers characterized and within therange observed for natural aptamers, and K_(D) values below most invitro selected aptamers characterized and within the range observed fornatural aptamers (FIG. 16). The slower k_(off) rates exhibited by thefolinic acid aptamers compared to other in vitro selected aptamers maybe a consequence of the increasing selection stringency applied throughextended buffer washing over the course of the selection. Throughcontrolling selection stringency, aptamers with high affinity andspecificity can be generated. Functional validation of their bindingincreases the confidence that these aptamers are capable of functioningin vivo.

TABLE 1 Kinetic and equilibrium binding properties of aptamers for (6R)-and (6S)-folinic acid. (6R)-Folinic Acid (6S)-Folinic Acid Aptamer k_(a)(M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M)Selectivity (6R)-FA tFA8-4 2.69E+05 5.15E−03 1.92E−08 2.11E+04 1.05E−014.98E−06 260 specific tFA8-11 1.16E+05 4.68E−03 4.05E−08 6.56E+032.27E−01 3.46E−05 855 tFA8-3 4.59E+05 2.17E−02 4.73E−08 — — 2.86E−05 606tFA6-8 7.79E+04 1.76E−02 2.25E−07 2.33E+03 2.97E−01 1.28E−04 567 tFA8-163.30E+04 6.02E−02 1.82E−06 6.82E+02 8.50E−01 1.25E−03 684 (6S)-FAtFA8-18 9.40E+01 3.55E−03 3.77E−05 3.61E+04 2.27E−03 6.28E−08 600specific tFA10-7 — — 7.44E−05 5.36E+04 7.75E−03 1.44E−07 515 tFA8-14 — —1.02E−02 8.92E+04 1.28E−02 1.44E−07 71,100

TABLE 2 Equilibrium binding properties of aptamers for folatederivatives. Tetrahydrofolic (6R)-Tetrahydro- Dihydrofolic acidbiopterin acid (THF) (THB) (DHF) Aptamer K_(D) (M) K_(D) (M) K_(D) (M)tFA8-4 (1.7-2.5)E−06 N.D.  (2.6-6.6)E−06 tFA8-11 (1.4-3.3)E−06 N.D.(7.2-19.7)E−06 tFA8-3 (2.2-2.6)E−06 1.6E−06  (1.1-3.5)E−06 tFA8-18 N.D.N.D. N.D. N.D. refers to non-detectable levels of binding at conditionstested.

Aptamer sequence-activity relationships identify suitable switchintegration site. Aptamer mutations were studied to assess their effecton ligand binding to better understand which nucleotides were involvedin or necessary for binding and to identify sequence flexibility thatcould be leveraged during switch design. In particular, integration intoregulatory switch platforms generally requires one or more stems throughwhich to couple the aptamer to the actuator. Thus, identifying potentialintegration stems whose sequence can be modified for facile switchintegration while not affecting ligand binding is a critical step inswitch design. Two predicted stems in the FAt8-4 truncated aptamer, thehighest affinity (6R)-specific aptamer identified, were assayed forsequence flexibility by shuffling nucleotide identify (A to C, C to A, Gto U, and U to G to maintain U-G base pairing) (FIG. 17). If shuffledstem sequences retained binding ability, stems were labeled as sequenceunconstrained without testing all six possible base pairs (i.e., A-U,U-A, C-G, G-C, U-G, G-U). Interestingly, one stem maintained bindingeven after shuffling all nucleotides, while extension of the other stemabolished binding activity. The sequence flexibility of the former stemprovides a suitable integration point for incorporation into regulatoryswitch platforms, including translation, splicing, RNA stability, andRNA interference-based mechanisms. The sequence constraints of thelatter stem suggest that either the predicted secondary structure isincorrect or that the terminal stem loop is involved in binding orfolding (FIG. 17). Homology also indicates that this sequence isconserved among multiple selected aptamers, consistent with its loss ofactivity when modified (FIG. 15).

With the identity of at most 23 nucleotides fixed, the FAt8-4 aptamerencodes extraordinarily high ligand affinity and specificity in a short,compact sequence. A means for quantifying and comparing the bindingcapacities of different aptamers is calculated through their informationcontent, defined as the amount of information (i.e., bits) necessary tospecify the identity of each nucleotide position. A simple measure ofinformation content adds two bits of information for invariantpositions, one bit for two-base varying positions such as base-pairednucleotides, and zero bits for unconstrained four-base varyingpositions. Thus, the information content of FAt8-4 has an upper limit ofapproximately 60 bits (23 invariant positions and 14 base-pairedpositions), with fewer base pairs likely required to stabilize the stemand possibly other positions that may not be entirely invariant.Compared to other characterized aptamers, the information contentnecessary to encode folinic acid binding is consistent with the fewother high-affinity aptamers analyzed.

Ribozyme-based switch transmitter screen in yeast. Syntheticribozyme-based switches have been shown to regulate gene expression in adose-dependent and reversible manner and exhibit a number of keyproperties: scalability, with their modular architecture allowinginterchangeable sensor and gene choice for programmable ligandinput-gene output combinations; tunability, with tunable gene-regulatoryactivities and directionality with switches either increasing ordecreasing gene expression in the presence of input ligand; andportability, with switches demonstrated in vitro and in viral,bacterial, fungal, and mammalian systems.

A transmitter library was screened for functional switches inSaccharomyces cerevisiae. An N11 transmitter library with 4.2 milliondevice variants was generated by randomizing 11 out of 15 positions in atransmitter component joining the folinic acid aptamer FAt8-4 to thesTRSV hammerhead ribozyme (FIG. 5a , FIG. 6). The first base pair oneither end of the transmitter was conserved from the FAt8-4 aptamer stemor from parent theophylline-responsive L2b8 switch, respectively. TheFAt8-4 aptamer was chosen due to its highest affinity among selected(6R)-specific folinic acid aptamers. Five sorts were performed (FIG. 5c), alternating between the absence and presence of 10 mM (6R,S)-folinicacid and using theophylline-responsive L2b8 in the absence (˜10.8%normalized GFP expression) and presence (˜36.7% normalized GFPexpression) of 5 mM theophylline, respectively, as the sortingcollection gates (FIG. 5b ).

Screening of isolated cells yielded folinic acid-responsive switcheswith activation ratios up to 6.1-fold in 3 mM (6R)-folinic acid (FIG.6), demonstrating aptamer activity in vivo. These switches maintainligand specificity for (6R)-folinic acid over (6S)-folinic acid in vivowith no induction of gene expression observed in the presence of(6S)-folinic acid. Both switches also achieve stringent regulatorysilencing in the absence of ligand as low as 3.4%, indicating that theyare biologically orthogonal and not sensitive to intracellular folatesor other metabolites or macromolecules, in contrast to some switchesthat have been developed. Interestingly, (6R)-FA-switch2 includes adeletion within the transmitter component, which only includes 14nucleotides. These switches demonstrate that selected folinic acidaptamers function intracellularly and can be coupled to ribozymes toconfer folinic acid-responsive gene regulation.

Switches may upregulate or downregulate gene expression in response toligand.

Switches may be present in different genetic contexts (e.g., differentpromoters, regulated genes, genomic integration vs. plasmid expression).

In vitro selection methods enable de novo generation of RNA sensingcapabilities, potentially allowing genetic switches to any desiredmolecular target. However, challenges associated with generating sensorcomponents that are amenable to subsequent switch integration and thatfunction in vivo have limited available sensing capabilities andpossible applications. By applying selection design principles thatfavor the selection of aptamers that bind with high affinity andspecificity under physiologically relevant conditions, a panel of RNAaptamers was generated that bind the clinical drug folinic acid withbinding affinities as low as 19.2 nM and specificities over 70,000-foldagainst structurally similar compounds. Through library screening,folinic acid-responsive RNA switches were engineered that activate geneexpression in vivo. Ribozyme-based switches have previously beendemonstrated to transfer from yeast or bacterial systems to mammaliansystems. In addition, the modular construction of these switches enablesfacile substitution of folinic acid aptamers into previously developedtheophylline-responsive switches implemented in clinically relevantproof-of-concept studies regulating T cell proliferation for adoptivetransfer therapy (see Chen et al. (2010) PNAS 107(19):8531-8536) andviral replication for gene therapy and vaccine development. The limitedtoxicity of folinic acid observed in clinical use indicates that thesesensing and regulatory capabilities are broadly applicable and provide ageneral, nontoxic small molecule effector for controlling geneexpression in bacterial, fungal, and mammalian systems and for moresensitive clinical applications. In addition to previously mentionedapplications in T cell proliferation and gene therapy, additionalpotential clinical applications include bacterial probiotic treatmentsincluding pathogen seeking and killing, tumor invasion, and livingdiagnostics.

The in vitro selected folinic acid aptamers can be compared to therecently discovered natural tetrahydrofolate riboswitch. Although alsoable to bind folinic acid, the tetrahydrofolate riboswitch exhibits muchlower affinity (˜10-20 μM), low specificity between diastereomers(<1.5-fold) and against purines (with certain purines even binding moretightly than the cognate ligand), long length (˜90 nt), and multipleligand binding sites, rendering it more challenging for incorporatinginto a synthetic regulatory switch and unsuitable for clinicalapplications due to its low specificity and affinity (Ames et al. (2010)Chemistry & Biology 17(7):681-685).

Applications for these aptamers include gene regulatory devices acrosscell and organism types and in therapeutic applications. In vivobiosensors for folate derivatives may be constructed from (6S)-folinicacid specific aptamers and used to monitor intracellular folatemetabolism, to engineer folate central metabolism of this criticalcofactor, or to engineer strains to overproduce these compounds. Theseaptamers can be evolved to bind to other folate analogues, both naturaland unnatural such as the antifolate drugs methotrexate or pemetrexed.The different pharmacokinetic properties of the two diastereomers offolinic acid enable switches with different temporal control to bedesigned: one with a fast-acting and short-lived pulse of geneexpression and a second with a longer, sustained pulse.

Aptamers and synthetic RNA regulatory switches provide a powerfulapproach for generating novel biological sensing and controlcapabilities with which to program gene expression and cellularbehavior. Coupling de novo, in vitro selection with regulatory switchconstruction methods holds promise for greatly expanding the ligandchemical diversity of inducible gene expression systems. Historically,not many aptamer selections have been conducted with the primary intentof in vivo function. To increase the likelihood of generating sensingcapabilities that function in vivo and are readily integrated intoregulatory switch platforms, a system of design, control, and validationwas applied to the process of generating a novel inducible geneexpression system. By rigorously controlling design parameters andquantitatively validating the processes of ligand immobilization,aptamer selection, aptamer screening, and switch construction, thissystem provides a strategy for identifying, prototyping, and carryingforward promising ligand, aptamer, and switch candidates and combinesthe strengths of in vitro and in vivo methods. This systematized processcan serve as a template and model to guide the development of otherRNA-based inducible gene regulatory systems and expand their possibleapplications in engineering increasingly sophisticated biologicalsystems.

TABLE 3 Selection Round 1 2 3 4 5 6 7 8 9 10 [FA] on Column 2 mM 2 mM200 μM 200 μM 20 μM 20 μM 2 μM 2 μM 2 μM 2 μM Buffer Wash 1 0.01% 0.02%0.00% 0.07% 0.06% 0.13% 0.02% 0.03% 0.06% 0.07% Buffer Wash 2 52.33% 58.84%  0.05% 0.07% 0.06% 0.13% 0.04% 0.03% 0.06% 0.28% Buffer Wash 342.16%  28.25%  28.01%  16.16%  17.64%  6.34% 24.63%  26.25%  16.88% 23.38%  Buffer Wash 4 4.36% 4.71% 50.03%  35.02%  50.41%  43.09% 47.01%  52.50%  51.93%  55.02%  Buffer Wash 5 0.44% 1.41% 16.51% 13.47%  18.90%  22.81%  22.39%  12.83%  18.18%  11.69%  Buffer Wash 60.17% 0.71% 2.50% 5.39% 6.30% 8.87% 2.69% 3.21% 2.99% 2.20% Buffer Wash7 0.42% 0.60% 4.71% 2.27% 4.06% 0.85% 0.88% 1.43% 1.03% Buffer Wash 80.33% 0.25% 3.37% 1.26% 2.53% 0.40% 0.58% 0.65% 0.55% Buffer Wash 90.24% 0.20% 3.03% 0.63% 1.77% 0.18% 0.47% 0.52% 0.41% Buffer Wash 100.15% 2.15% 0.38% 1.27% 0.13% 0.29% 0.45% 0.34% Buffer Wash 11 2.02%0.32% 1.27% 0.09% 0.23% 0.39% 0.34% Buffer Wash 12 1.75% 0.19% 0.76%0.07% 0.23% 0.32% 0.28% Buffer Wash 13 1.48% 0.19% 0.63% 0.06% 0.18%0.32% 0.28% Buffer Wash 14 0.51% 0.06% 0.16% 0.32% 0.21% Buffer Wash 150.51% 0.04% 0.15% 0.26% 0.21% Buffer Wash 16 0.51% 0.04% 0.15% 0.32%0.21% Buffer Wash 17 0.12% 0.26% 0.21% Buffer Wash 18 0.12% 0.19% 0.21%Buffer Wash 19 0.09% 0.19% 0.14% Buffer Wash 20 0.14% Buffer Wash 210.14% Buffer Wash 22 0.14% Sum Buffer 99.48%  94.93%  98.30%  88.69% 98.61%  95.18%  98.70%  98.48%  95.75%  97.46%  Washes [FA] in Elutions20 mM 20 mM 20 mM 20 mM 2 mM 2 mM 200 μM 200 μM 200 μM 200 μM FA Elution1 0.12% 0.19% 0.15% 1.35% 0.19% 0.51% 0.04% 0.09% 0.23% 0.14% FA Elution2 0.17% 0.38% 0.30% 8.08% 0.38% 2.28% 0.09% 0.20% 1.17% 0.62% FA Elution3 0.03% 0.14% 0.15% 0.81% 0.13% 0.51% 0.04% 0.09% 0.45% 0.21% FA Elution4 0.03% 0.06% 0.10% 0.20% 0.06% 0.25% 0.07% 0.18% 0.91% 0.41% FA Elution5 0.01% 0.05% 0.05% 0.13% 0.06% 0.13% 0.04% 0.09% 0.32% 0.14% FA Elution6 0.01% 0.02% 0.05% 0.07% 0.06% 0.13% 0.02% 0.06% 0.13% 0.07% Sum FA0.38% 0.84% 0.80% 10.64%  0.88% 3.80% 0.31% 0.70% 3.21% 1.58% ElutionsBlocked — — 0.60% 0.40% 0.32% 0.63% 0.81% 0.53% 0.78% 0.76% NegativeSelection Column FA Conjugated 0.15% 4.24% 0.30% 0.27% 0.19% 0.38% 0.18%0.29% 0.26% 0.21% Column In vitro selection design, stringency, andprogress. Steady increases in selection stringency are incorporated intoselection through gradual increases in buffer wash volume, decreases inconcentration of folinic acid conjugated onto solid support beads, anddecreases in concentration of folinic acid in elution. Selectionprogress is monitored through RNA retention profile. Percentage of³²P-radiolabeled RNA in each fraction is measured using a Geigercounter. Volume of all columns, buffer washes, and elutions is 0.5 mL.Blocked negative selections columns are used in selection rounds 3through 10. Input RNA library is added to negative selection column andflows into folinic acid conjugated selection column with addition ofbuffer washes, shifting RNA retention profile by one wash (i.e., peakRNA fractions are buffer washes 2 and 3 for rounds 1 and 2 but shifts tobuffer washes 3 and 4 for remaining rounds since RNA must travel throughan additional volume of beads in second column). Negative selectioncolumns are removed after four buffer washes, and remaining bufferwashes and elutions are added directly to selection column.

TABLE 4 Sequences of folinic acid aptamers. In vitro selectionAntisense DNA sequence N70 library5′-GTGTCCGCCTATCTCGTCTCCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN SEQ ID NO: 1NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGGAAGGAGGCGGGCAGAAGTCCCTATAGTGAGTCGTATTAGAA AptamersFull-length RNA sequence from in vitro selection (6R)-FA specificaptamers FA8-4 5′-GGGACUUCUGCCCGCCUCCUUCCUGCUCGUGUCAAAAUGAAUGGCGCUCGGCGUSEQ ID NO: 2 UGCGUGGUACGUUAUAUUCCGGCCAAGCAGCCAUUCAUGGGAGACGAGAUAGGCGGACAC FA8-3 5′-GGGACUUCUGCCCGCCUCCUUCCGCUGAGGACUCGGCACCGAAUUUGCCAACGUSEQ ID NO: 3 CUGGUCACGACCGUAGUACACUACCCCUCGAAAUCACGAGGGAGACGAGAUAGGCGGACAC FA8-11 5′-GGGACUUCUGCCCGCCUCCUUCCGCUUACCGGACGCCUUAAGGCAUCAGCAUGCSEQ ID NO: 4 AGUGCUUGGUACGUUAUAUUCAGCUGCAACUCGGGAUGCGGAGACGAGAUAGGCGGACAC FA6-8 5′-GGGACUUCUGCCCGCCUCCUUCCGCUAUAAGUCGGACUUCCCGUCAGGUACGUUSEQ ID NO: 5 AUAUUCGGGGGAGUACGGUUAAAGCAGAUUUGUUGAAUGGAGACGAGAUAGGCGGACAC FA8-16 5′-GGGACUUCUGCCCGCCUCCUUCCGGUGACCUGGACGUUAUUUCCGGCCGAAGGGSEQ ID NO: 6 AGACGAGAUAGGCGGACAC (6S)-FA specific aptamers FA8-185′-GGGACUUCUGCCCGCCUCCUUCCUACUCCCGACUCGUCAAUGCAUAUGUAACGU SEQ ID NO: 7UAACGGCGCUACCUGUACAAUGCACGUUCCGAGCGCCCGGAGACGAGAUAGGCG GACAC FA10-75′-GGGACUUCUGCCCGCCUCCUUCCCCGACACGGCGAAGAGUCAAAGCAUCCCCUG SEQ ID NO: 8CAUGGAGCCAACAAGCCCUGCCUCCACGCAGGGCCCGUGGGAGACGAGAUAGGC GGACAC FA8-145′-GGGACUUCUGCCCGCCUCCUUCCGCGCAAGUGCAUUAACGGUGACACCGAAAGC SEQ ID NO: 9UGGAAAGCCUGACAUCGAACGCAAAAGGCUGCGUGGCAUGGAGACGAGAUAGGC GGACAC TruncatedAptamers DNA sequence for SPR (6R)-FA specific aptamers FAt8-45′-TTCTAATACGACTCACTATAGGGGCTTGGCGTTGCGTGGTACGTTATATTCCGG SEQ ID NO: 10CCAAGCCCCCAAAAAAAAAAAAAAAAAAAAAAAA FAt8-35′-TTCTAATACGACTCACTATAGGGTTTGCCAACGTCTGGTCACGACCGTAGTACA SEQ ID NO: 11CTACCCCTCGAAATCACGAGGGAGACGAGATAGGCGGACCCCAAAAAAAAAAAA AAAAAAAAAAAAFAt8-11 5′-TTCTAATACGACTCACTATAGGGTGCAGTGCTTGGTACGTTATATTCAGCTGCASEQ ID NO: 12 CCCCAAAAAAAAAAAAAAAAAAAAAAAA FAt6-85′-TTCTAATACGACTCACTATAGGGTTCCCGTCAGGTACGTTATATTCGGGGGACC SEQ ID NO: 13CCAAAAAAAAAAAAAAAAAAAAAAAA FAt8-165′-TTCTAATACGACTCACTATAGGGCTCCTTCCGGTGACCTGGACGTTATTTCCGG SEQ ID NO: 14CCGAAGGGAGCCCCAAAAAAAAAAAAAAAAAAAAAAAA (6S)-FA specific aptamers FAt8-185′-TTCTAATACGACTCACTATAGGGCTCGTCAATGCATATGTAACGTTAACGGCGC SEQ ID NO: 15TACCTGTACAATGCACGTTCCGAGCGCCCGGAGACGAGCCCCAAAAAAAAAAAA AAAAAAAAAAAAFAt10-7 5′-TTCTAATACGACTCACTATAGGGACGGCGAAGAGTCAAAGCATCCCCTGCGAAASEQ ID NO: 16 GCAGGGCCCGTCCCCAAAAAAAAAAAAAAAAAAAAAAAA FAt8-145′-TTCTAATACGACTCACTATAGGGCCGCGCAAGTGCATTAACGGTGACACCGAAA SEQ ID NO: 17GCTGGAAAGCCTGACATCGAACGCAAAAGGCTGCGTGGCCCCAAAAAAAAAAAA AAAAAAAAAAAATruncated Aptamers RNA sequence (6R)-FA specific aptamers FAt8-45′-GCUUGGCGUUGCGUGGUACGUUAUAUUCCGGCCAAGC SEQ ID NO: 18 FAt8-35′-UUUGCCAACGUCUGGUCACGACCGUAGUACACUACCCCUCGAAAUCACGAGGGA SEQ ID NO: 19GACGAGAUAGGCGGA FAt8-11 5′-UGCAGUGCUUGGUACGUUAUAUUCAGCUGCA SEQ ID NO: 20FAt6-8 5′-UUCCCGUCAGGUACGUUAUAUUCGGGGGA SEQ ID NO: 21 FAt8-165′-CUCCUUCCGGUGACCUGGACGUUAUUUCCGGCCGAAGGGAG SEQ ID NO: 22(6S)-FA specific aptamers FAt8-185′-CUCGUCAAUGCAUAUGUAACGUUAACGGCGCUACCUGUACAAUGCACGUUCCGA SEQ ID NO: 23GCGCCCGGAGACGAG FAt10-7 5′-ACGGCGAAGAGUCAAAGCAUCCCCUGCGAAAGCAGGGCCCGUSEQ ID NO: 24 FAt8-145′-CCGCGCAAGUGCAUUAACGGUGACACCGAAAGCUGGAAAGCCUGACAUCGAACG SEQ ID NO: 25CAAAAGGCUGCGUGG Mutated Aptamer DNA sequence for SPR FAt8-4-stem15′-TTCTAATACGACTCACTATAGGGTAGGTTCGTTGCGTGGTACGTTATATTCCGG SEQ ID NO: 26AACCTACCCCAAAAAAAAAAAAAAAAAAAAAAAA FAt8-4-stem25′-TTCTAATACGACTCACTATAGGGTAGGTTAGTTGCGTGGTACGTTATATTCCGT SEQ ID NO: 27AACCTACCCCAAAAAAAAAAAAAAAAAAAAAAAA FAt8-4-stem35′-TTCTAATACGACTCACTATAGGGGCTTGGCGTTGCGTGCAGTGAGCCTACTGGT SEQ ID NO: 28ACGTTATATTCCGGCCAAGCCCCCAAAAAAAAAAAAAAAAAAAAAAAA FAt8-4-stem45′-TTCTAATACGACTCACTATAGGGGCTTGGCGTTGCGTGGCAGTGAGCCTACTGT SEQ ID NO: 29ACGTTATATTCCGGCCAAGCCCCCAAAAAAAAAAAAAAAAAAAAAAAA Primers DNA sequenceN70-fwd 5′-TTCTAATACGACTCACTATAGGGACTTCTGCCCGCCTCCTTCC SEQ ID NO: 30N70-fwd-short 5′-TTCTAATACGACTCACTATAGGGACTTCTGCCCGCCTC SEQ ID NO: 31N70-rev 5′-GTGTCCGCCTATCTCGTCTCC SEQ ID NO: 32 GAP-N70-AvrII-fwd-5′-TCCATGGTATGGATGAATTGTACAAATAAAGCCTAGGGGGACTTCTGCCCGCCT short CSEQ ID NO: 33 GAP-N70-XhoI-rev5′-AAGAAATTCGCTTATTTAGAAGTGGCGCGCCCTCTCGAGAGTGTCCGCCTATCT SEQ ID NO: 34CGTCTCC Biacore-N70-rev5′-TTTTTTTTTTTTTTTTTTTTTTTTGGGGGTGTCCGCCTATCTCGTCTCC SEQ ID NO: 35Biacore-fwd 5′-TTCTAATACGACTCACTATAGGG SEQ ID NO: 36 Biacore-rev5′-TTTTTTTTTTTTTTTTTTTTTTTTGGGG SEQ ID NO: 37

TABLE 5Sequences for transmitter-based device library and isolated folinicacid-responsive switches. RNA Switches DNA sequence FA8-4-sTRSV N115′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGOTCTGATGAGTCCGTTNNNCGTTtransmitter libraryGCGTGGTACGTTATATTCCOGNNNNNNNNGGAGGACGAAACAGCAAAAAGAAAAATA SEQ ID NO: 38AAAA (6R)-FA-switch15′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGOTCTGATGAGTCCGTTGGTCGTTSEQ ID NO: 39 GCGTGGTACGTTATATTCCOGGCCGAACGGGAGGACGAAACAGCAAAAAGAAAAATAAAAA (6R)-FA-switch25′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGOTCTGATGAGTCCGTTGCTCGTTSEQ ID NO: 40 GCGTGGTACGTTATATTCCOGGGCGAACGGAGGACGAAACAGCAAAAAGAAAAATAAAAA sTRSV 5′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGOTCTGATGAGTCCGTGAGGACGASEQ ID NO: 41 AACAGCAAAAAGAAAAATAAAAA sTRSV Contl5′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTACGTGAGGTCCGTGAGGACAGSEQ ID NO: 42 AACAGCAAAAAGAAAAATAAAAA L2b85′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGOTCTGATGAGTCCGTTGTCCATASEQ ID NO: 43 CCAGCATCGTCTTGATGCCCTTGGCAGGGACGGGACGGAGGACGAAACAGCAAAAAGAAAAATAAAAA Primers DNA sequence GAP-N70-AvrII-fwd-5′-TCCATGGTATGGATGAATTGTACAAATAAAGCCTAGGGGGACTTCTGCCCGCCTC shortSEQ ID NO: 44 GAP-N70-XhoI-rev5′-AAGAAATTCGCTTATTTAGAAGTGGCGCGCCCTCTCGAGAGTOTCCGCCTATCTCGTSEQ ID NO: 45 CTCC GAP-L1-2-fwd5′-TCCATGGTATGGATGAATTGTACAAATAAAGCCTAGGAAACAAACAAAGCTGTCACCSEQ ID NO: 46 GAP-L1-2-rev5′-AAGAAATTCGCTTATTTAGAAGTGGCGCGCCCTCTCGAGTTTTTATTTTTCTTTTTGSEQ ID NO: 47 CTGTTTCG L1-2-fwd 5′-GACCTAGGAAACAAACAAAGCTGTCACCSEQ ID NO: 48 L1-2-rev 5′-GGCTCGAGTTTTTATTTTTCTTTTTGCTGTTTCGSEQ ID NO: 49 CS653 5′-GGTCACAAATTGGAATACAACTATAACTCT SEQ ID NO: 50CS654 5′-CGGAATTAACCCTCACTAAAGGG SEQ ID NO: 51 Plasmids Plasmid NumbersTRSV pCS1750 sTRSV Contl pCS1751 GFP pCS1585 mCherry pCS1749 no colorpCS4 L2b8 pCS1753 GFP-mCherry pCS1748

Example 2

Aptamer mutations were studied to assess their effect on ligand bindingto better understand which nucleotides were involved in or necessary forbinding and to identify sequence flexibility that could be leveragedduring switch design. In particular, integration into regulatory switchplatforms generally requires one or more stems through which to couplethe aptamer to the actuator. Thus, identifying potential integrationstems whose sequence can be modified for facile integration while notaffecting ligand binding is a critical step in switch design.

Aptamer stems were assayed for sequence flexibility or constraints byshuffling nucleotide identify (A to C, C to A, G to U, and U to G tomaintain U-G basepairing). If shuffled stem sequences retained bindingability, stems were labeled as sequence unconstrained without testingall six possible base pairs (A-U, U-A, C-G, G-C, U-G, G-U). Terminalstem loop lengths were tested to minimize stem length, and individualpoint mutations were rationally picked and tested (FIG. 18). Aptamersspecific for both (6R)- and (6S)-folinic acid were identified thatcontained two sequence unconstrained stems, thus enabling aptamerintegration into regulatory platforms requiring either one (e.g.,ribozyme-based) or two (e.g., miRNA or Rnt1p-based) integration sites.Aptamers with more than one integration stem provide more designflexibility even for switch platforms only requiring one site, asaptamer orientation can be chosen.

Next, particular functional groups of folinic acid that were importantfor aptamer binding were tested. With many natural and unnatural folateanalogues available, binding of two derivatives were characterized thataltered specific functionalities of folinic acid, which is composed ofpterin, para-aminobenzoate, glutamate, and formyl moieties (FIG. 19).(6R,S)-5-methyl-5,6,7,8-tetrahydrofolic acid replaces the 5-formyl groupwith a 5-methyl group, testing the role of the formyl oxygen on binding.(6R,S)-5-formyl-5,6,7,8-tetrahydropteroic acid removes the glutamategroup through hydrolysis of the amide bond. While bindingcharacterization indicates a significant role of the 5-formyl oxygen inbinding, the glutamate residue appears to be nonessential (Table 6).Removing the glutamate residue maintains or potentially increasesbinding affinity, possibly due to the decrease in conformationalflexibility of the ligand from the number of rotatable bonds, decreasingentropic cost of ligand binding necessary for locking molecule into arigid binding conformation, and possibly preventing the glutamate moietyfrom folding into a conformation that prevents or disrupts binding withRNA. By removing the unnecessary moiety, the kinetics of binding (k_(a))are potentially increased.

TABLE 6 (6R,S)-5-methyl-THF (6R,S)-5-formyl-THPteroic Acid Aptamer k_(a)(M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M)  8-43.35E+04 2.11E−01 6.29E−06 1.35E+05 4.94E−03 3.66E−08  8-11 1.43E+041.95E−01 1.36E−05 7.50E+05 5.68E−03 7.57E−08  8-3 — — 1.58E−07 3.05E+052.92E−02 9.57E−08  8-18 — — 4.47E−04 5.32E+04 6.33E−03 1.19E−07 10-7 — —8.18E−05 3.52E+04 6.97E−03 1.98E−07 Kinetic and equilibrium bindingproperties of aptamers for folinic acid derivatives. (6R,S)-5-methyl-THFand (6R,S)-5-formyl-THPteroic acid are mixtures of both (6R) and (6S)diastereomers; thus, when comparing binding to the specific folinic aciddiastereomer, k_(a) and K_(D) values should be adjusted accordingly.

More detailed structural information on aptamer-ligand binding can beobtained through chemical probing experiments such as SHAPE,crystallography, or nuclear magnetic resonance imaging. More detailedinformation on the energy binding landscape could be obtained throughsingle-molecule force and folding studies or high-throughput mutationaland affinity measurements such as RNA-MITOMI. In addition, since theglutamate moiety of folinic acid is not necessary for ligand binding,tagged derivatives of folinic acid can be synthesized, using theglutamate residue of folinic acid or the benzoic acid of5-formyl-5,6,7,8-tetrahydropteoric acid as a chemical handle to couple acargo such as a fluorophore tag to. The availability of diverse folateanalogues can also be mined to test the effect of different oxidationstates of the carbon atoms in the 5, 6, 7, or 8 positions or ligandconformation through derivatives that bridge the 5′ and 10′ nitrogens.

Applications for these aptamers include gene-regulatory devices acrosscell and organism types and in therapeutic applications. In vivobiosensors for folate derivatives may be constructed from (6S)-folinicacid specific aptamers and used to monitor intracellular folatemetabolism, to engineer folate central metabolism of this criticalcofactor, or to engineer strains to overproduce these compounds. Theseaptamers may be evolved to bind to other folate analogues, both naturaland unnatural such as the antifolates methotrexate or pemetrexed.

Example 3

Rational design of ribozyme-based switches in yeast. Folinicacid-responsive switches were first rationally designed to demonstrateaptamer in vivo activity. The strand displacement mechanism previouslyused to rationally design theophylline- and tetracycline-responsiveswitches was tested on two different switch architectures: aptamerintegration off of the hammerhead loop II and integration throughhammerhead helix III (FIG. 20). For loop II integration, transmittersequences from previously characterized switches were used as a startingpoint to join folinic acid aptamers FA8-4 and FA8-3 to the sTRSVhammerhead ribozyme, tested in silico using RNA folding programs, andmodified as necessary to achieve proper folding of the ribozyme andaptamer domains.

Helix III integration represents an alternate switch architecture thathas not previously been reported. Aptamer integration through helix IIIrequires two integration stems on the aptamer: one for ribozyme helixIII integration and another for transcript integration. In thisarchitecture, the ribozyme sequence does not need to be altered, andtherefore the native tertiary loop-loop interactions are maintained,with the goal of achieving lower basal activity and eliminating the needto rescue impaired tertiary interactions. However, this integrationmethod could potentially be kinetically unfavorable, as the entireribozyme sequence is transcribed before the aptamer is, possiblyreducing the time for ligand binding to prevent ribozyme cleavage. Thisintegration design is partially inspired by reported in vitro aptamericsensors that couple two aptamer domains, in vivo miRNA and Rnt1p switchdesigns, and Spinach-based sensors. However, these sensors and switchesoperate through conformational changes upon ligand binding thatstabilize connecting stems (e.g., communication modules) or sequesteraptamer nucleotides from enzymatic recognition and processing, thusmaking the use of a transmitter junction a novel switch architecture.

Switches achieved activation ratios up to 2.4-fold, while retainingspecificity for (6R)-folinic acid over (6S)-folinic acid (FIG. 21).These switches demonstrate that selected folinic acid aptamers functionintracellularly and can be coupled to ribozymes to confer folinicacid-responsive gene regulation. Of interest to note is that ribozymeactive structure for FA8-3-switch9 is the second lowest energy predictedstructure, in contrast to most designed ribozyme-based switches, whereit is the lowest energy structure (−44.7 kcal/mol vs. −45.6 kcal/mol foraptamer folded structure).

Example 4 Ribozyme Loop Replacement Screen in Yeast

An alternative switch architecture based on ribozyme loop replacementhas recently been demonstrated to generate theophylline-responsiveswitches that function in vivo. This strategy relies on the observationthat natural hammerhead ribozymes possess many sequence solutions formaintaining tertiary loop-loop interactions that are crucial forstringent regulatory silencing (FIG. 22). Previous work has also enabledimpaired loop-loop interactions to be rescued through screening of acompletely randomized ribozyme loop, resulting in lower basal activity.Combining these two observations, this integration strategy replaces oneof the two interacting hammerhead loops with an aptamer, placing aninternal or terminal loop of an aptamer approximately in the sameposition as the replaced ribozyme loop. To rescue the loop-loopinteraction, the second loop is completely randomized and the devicelibrary is screened for functional switches. In the absence of ligand,nucleotides of the two loops are predicted to interact through tertiaryinteractions. However, in the presence of ligand, ligand binding to theaptamer sequesters aptamer nucleotides involved in the loop-loopinteraction, precluding proper tertiary contact formation and disruptingribozyme cleavage. Because refolding of the aptamer domain isunnecessary in this switch architecture, this switch design has beenshown to be more sensitive than transmitter-based designs, resulting ina lower IC50 needed to activate the switch. Previously designedtransmitter-based ON switches require two functional conformations: onewith active ribozyme and inactive aptamer and a second with a properlyfolded aptamer and an improperly folded ribozyme. These two functionalconformations are generally among the lowest energy predicted secondarystructures, with a slightly energetically favored active ribozymeconformation and with a difference in energy small enough for ligandbinding to favor the active aptamer conformation. With both the ribozymeand aptamer folded into their functional conformations, removingcompeting secondary structures should also lead to lower basalactivities (assuming that tertiary contacts can be restored) and canavoid impairing aptamer affinities upon switch integration that havebeen previously observed.

Fourteen device libraries based on ribozyme loop replacement weresynthesized and pooled together for cell sorting (FIG. 23). Two folinicacid aptamers, FA8-4 and FA8-11, were chosen for their high affinity,short lengths, and unconstrained base stems and replaced either loop Ior loop II in device libraries. Library designs were inspired bysequence diversity of natural hammerhead ribozymes, with prevalentcombinations of stem and loop lengths used as the basis of designedlibraries. Positions within the aptamer domain that had been identifiedas variable were also randomized. These positions, located near the basestem of the aptamer, were hypothesized to occupy loop positions thatwould be more likely to be involved in forming tertiary contacts; thus,additional diversity in these privileged positions would potentiallylead to functional switches. For switches including aptamer FA8-4, thebase stem sequence was derived from the parent ribozyme. For switchesincluding aptamer FA8-11, the first base pair of the base stem sequencewas conserved as a U-G base pair as this base pair was observed to beconserved in the functional aptamer.

Four rounds of cell sorting were conducted, using sorting collectiongates based off of two previously characterized theophylline switcheswith activities of ˜3.5% for the negative sorting gate in the absence offolinic acid and ˜36.7% for the positive sorting gate in the presence offolinic acid (FIG. 24a ).

Isolated cells were screened for switching activity, sequenced, andre-cloned from synthesized oligonucleotides into a fresh plasmidbackbone for characterization. Interestingly, only a single switch,FA-22, with an aptamer replacing loop II of the ribozyme was identified.This lack of functional solutions with aptamers replacing loop II couldreflect fewer natural examples of hammerhead ribozymes with a largerloop II relative to loop I. In addition, aptamer integration within loopI may be kinetically favored, as the aptamer would be fully transcribedbefore the ribozyme, providing more time for ligand binding beforeribozyme cleavage. As the sorts were designed to isolated functionalswitches and not solely functional ribozymes, it is possible thataptamer integration in loop II could produce active ribozymes but wereunfavorable switches compared to loop I integration. Three otherfamilies of switches were identified (FIG. 26). The highest performingswitches demonstrated up to 25.2-fold activation ratios (ratio of GFPexpression in the presence of ligand to expression in the absence ofligand) in the presence of 3 mM (6R)-folinic acid and dynamic ranges ofup to 43.6% (difference between GFP expression in the presence andabsence of ligand).

More switches incorporating aptamer FA8-11 were isolated. One hypothesisis that aptamer FA8-11 may facilitate greater conformational changesupon ligand binding. The required U-G base pair in this aptamer isnecessary for ligand binding; these two nucleotides are also present inaptamer FA8-4 but not contained within a base stem. This base pair,while base paired in the absence of ligand, may open up and formcontacts with the ligand upon binding. If true, ligand binding would notonly cause local conformation changes within the aptamer but would alsodisrupt a base pair in a stem of the ribozyme, further impairingtertiary interactions between the two ribozyme loops. This hypothesiscan be tested using chemical probing techniques such as SHAPE to assesschanges in RNA structure in the absence and presence of folinic acid.

Example 5 Rational Design of microRNA-Based Switches in Mammalian Cells

Ligand-responsive, microRNA (miRNA) switches have been developed thatmodulate Drosha processing. These switches integrate an aptamer into thebasal segments of a miRNA. Internal loop size contained within the basalsegments affects Drosha processing and therefore the levels ofmiRNA-mediated gene silencing. By integrating an aptamer within thebasal segments, unbound aptamer can remain relatively unstructured,while ligand binding can sequester nucleotides involved in binding andinhibit Drosha processing.

Aptamer integration into this switch platform requires two integrationstems; therefore, aptamer FA8-3 was chosen. Separate studies on usingthis aptamer in a transmitter-based switch observe that an internal loopwithin the aptamer could be expanded while retaining ligand bindingability. This loop was expanded to the previously identified optimalloop size and integrated into the miRNA switch that targets GFP. Asingle copy of the miRNA switch enables up to 2.1-fold change in GFPexpression (FIG. 26b ); including two copies yields up to 2.6-foldchange (FIG. 26c ). Additional copies of the switch would be expected tofurther increase the achievable activation ratio, as would specific useof (6R)-folinic acid.

TABLE 7Sequences for rationally designed and miRNA-based folinic acid-responsiveswitches and for device libraries based on ribozyme loop replacementstrategy and isolated folinic acid-responsive switches. Rationallydesigned switches DNA sequence FA8-4-switch45′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTTGCTGGCGTTSEQ ID NO: 52GCGTGGTACGTTATATTCCGGCCAGTTCAGCGGAGGACGAAACAGCAAAAAGAAAAATA AAAAFA8-3-switch95′-AAACAAACAAATTTGCCAACGTCTGGTCACGACCGCTGTCACCGGATGTGCTTTCCGGTSEQ ID NO: 53CTGATGAGTCCGTGAGGACGAAACAGCACAGCCCCTCGAAATCACGAGGGAGACGAGATAGGCGGAAAAAAGAAAAATAAAAA 8-3-switch15′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTTGTCCCAACGSEQ ID NO: 54TCTGGTCACGACCGTAGTACACTACCCCTCGAAATCACGAGGGAGACGAGATAGGGACGGGACGGAGGACGAAACAGCAAAAAGAAAAATAAAAA 8-3-switch35′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTTGAGTCAACGSEQ ID NO:55 TCTGGTCACGACCGTAGTACACTACCCCTCGAAATCACGAGGGAGACGAGATAGACTGATTACGGAGGACGAAACAGCAAAAAGAAAAATAAAAA 8-3-switch45′-AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTTGCTGCAACGSEQ ID NO: 56TCTGGTCACGACCGTAGTACACTACCCCTCGAAATCACGAGGGAGACGAGATAGCAGTTCAGCGGAGGACGAAACAGCAAAAAGAAAAATAAAAA miRNA-based switches DNA sequenceFA8-3-miRNA5′-AATAACCGGTGCGATCGCGAACGGGTCCTCACTACCCCTCGAAATCACGAGGGAGACGASEQ ID NO: 57GATAAGAGCGACGGCGGAGCGAGCACAAGCTGGAGTACAACTATAGTGAAGCCACAGATGTATAGTTGTACTCCAGCTTGTGCCCGCCTTGCCAACCATAACGTCTGGTCACGACCGTAGTAAGGGCCCGTTTTTAATTAAATCGATTATT Device libraries Sequence6bpL1N6/4bpL2FA8-4 CCTAGGAAACAAACAAAGCTGTCACCGGANNNNNNTCCGGTCTGATGAGTCSEQ ID NO: 58 CGNTGCGTGGTACGTTATATTCCGGGACGAAACAGCAAAAAGAAAAATAAAAACTCGAG 6bpL1N7/4bpL2FA8-4CCTAGGAAACAAACAAAGCTGTCACCGGANNNNNNNTCCGGTCTGATGAGT SEQ ID NO: 59CCGNTGCGTGGTACGTTATATTCCGGGACGAAACAGCAAAAAGAAAAATAA AAACTCGAG6bpL1FA8-4/3bpL2N6 CCTAGGAAACAAACAAAGCTGTCACCGGAGNTGCGTGGTACGTTATATTCCSEQ ID NO: 60 GTCCGGTCTGATGAGTCNNNNNNGACGAAACAGCAAAAAGAAAAATAAAAA CTCGAG6bpL1FA8-4/3bpL2N7 CCTAGGAAACAAACAAAGCTGTCACCGGAGNTGCGTGGTACGTTATATTCCSEQ ID NO: 61 GTCCGGTCTGATGAGTCNNNNNNNGACGAAACAGCAAAAAGAAAAATAAAAACTCGAG 6bpL1FA8-4/4bpL2N6CCTAGGAAACAAACAAAGCTGTCACCGGAGNTGCGTGGTACGTTATATTCC SEQ ID NO: 62GTCCGGTCTGATGAGTCCNNNNNNGGACGAAACAGCAAAAAGAAAAATAAA AACTCGAG6bpL1N6/4bpL2FA8-11 CCTAGGAAACAAACAAAGCTGTCACCGGANNNNNNTCCGGTCTGATGAGTCSEQ ID NO: 63 TGCNTGGTACGTTATATTCRGGACGAAACAGCAAAAAGAAAAATAAAAACT CGAG6bpL1N7/4bpL2FA8-11 CCTAGGAAACAAACAAAGCTGTCACCGGANNNNNNNTCCGGTCTGATGAGTSEQ ID NO: 64 CTGCNTGGTACGTTATATTCRGGACGAAACAGCAAAAAGAAAAATAAAAAC TCGAG6bpL1FA8-11/3bpL2N6 CCTAGGAAACAAACAAAGCTGTCACCGGTGCNTGGTACGTTATATTCRGCCSEQ ID NO: 65 GGTCTGATGAGTCNNNNNNGACGAAACAGCAAAAAGAAAAATAAAAACTCG AG6bpL1FA8-11/3bpL2N7 CCTAGGAAACAAACAAAGCTGTCACCGGTGCNTGGTACGTTATATTCRGCCSEQ ID NO: 66 GGTCTGATGAGTCNNNNNNNGACGAAACAGCAAAAAGAAAAATAAAAACTC GAG6bpL1FA8-11/4bpL2N6 CCTAGGAAACAAACAAAGCTGTCACCGGTGCNTGGTACGTTATATTCRGCCSEQ ID NO: 67 GGTCTGATGAGTCCNNNNNNGGACGAAACAGCAAAAAGAAAAATAAAAACT CGAG5bpL1FA8-4/3bpL2N7 CCTAGGAAACAAACAAAGCTGTCACCGGGNTGCGTGGTACGTTATATTCCGSEQ ID NO: 68 CCGGTCTGATGAGTCNNNNNNNGACGAAACAGCAAAAAGAAAAATAAAAAC TCGAG3bpL1N7/5bpL2FA8-4 CCTAGGAAACAAACAAAGCTGTCACCNNNNNNNGGTCTGATGAGTCCCGNTSEQ ID NO: 69 GCGTGGTACGTTATATTCCGGGGACGAAACAGCAAAAAGAAAAATAAAAAC TCGAG5bpL1FA8-11/3bpL2N7 CCTAGGAAACAAACAAAGCTGTCACCGTGCNTGGTACGTTATATTCRGCGGSEQ ID NO: 70 TCTGATGAGTCNNNNNNNGACGAAACAGCAAAAAGAAAAATAAAAACTCGA G3bpL1N7/5bpFA8-11 CCTAGGAAACAAACAAAGCTGTCACCNNNNNNNGGTCTGATGAGTCCTGCNSEQ ID NO: 71 TGGTACGTTATATTCRGGGACGAAACAGCAAAAAGAAAAATAAAAACTCGA GLoop replacement switch Sequence FA-4AAACAAACAAAGCTGTCACCGGTGCATGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 72ATGAGTCCTGGGGGGACGAAACAGCAAAAAGAAAAATAAAAA FA-6AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 73ATGAGTCTTGAGGAGACGAAACAGCAAAAAGAAAAATAAAAA FA-7AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 74ATGAGTCCACAGAGGGACGAAACAGCAAAAAGAAAAATAAAAA FA-9AAACAAACAAAGCTGTCACCGGAGATGCGTGGTACGTTATATTCCGTCCGG SEQ ID NO: 75TCTGATGAGTCCAGAGGGACGAAACAGCAAAAAGAAAAATAAAAA FA-11AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 76ATGAGTCTTGGAGAGACGAAACAGCAAAAAGAAAAATAAAAA FA-12AAACAAACAAAGCTGTCACCGGAGTTGCGTGGTACGTTATATTCCGTCCGG SEQ ID NO: 77TCTGATGAGTCCAAAGGGGACGAAACAGCAAAAAGAAAAATAAAAA FA-14AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 78ATGAGTCTTGAAGAGACGAAACAGCAAAAAGAAAAATAAAAA FA-18AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 79ATGAGTCTTCAAGAGACGAAACAGCAAAAAGAAAAATAAAAA FA-19AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 8-ATGAGTCCAAAGGGGGACGAAACAGCAAAAAGAAAAATAAAAA FA-22AAACAAACAAAGCTGTCACCGGAGGTGTAGTCCGGTCTGATGAGTCCGTTG SEQ ID NO: 81CGTGGTACGTTATATTCCGGGACGAAACAGCAAAAAGAAAAATAAAAA FA-27AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 82ATGAGTCCTGGAGGGACGAAACAGCAAAAAGAAAAATAAAAA FA-32AAACAAACAAAGCTGTCACCGGAGTTGCGTGGTACGTTATATTCCGTCCGG SEQ ID NO: 83TCTGATGAGTCTAGAAGAGACGAAACAGCAAAAAGAAAAATAAAAA FA-34AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 84ATGAGTCTTAAAGAGACGAAACAGCAAAAAGAAAAATAAAAA FA-58AAACAAACAAAGCTGTCACCGGTGCTTGGTACGTTATATTCAGCCGGTCTG SEQ ID NO: 85ATGAGTCCATAGAGGGACGAAACAGCAAAAAGAAAAATAAAAA Ribozyme controls SequencesTRSV Contl AAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTACGTGAGGTCCGTGASEQ ID NO: 86 GGACAGAACAGCAAAAAGAAAAATAAAAA sTRSVAAACAAACAAAGCTGTCACCGGATGTGCTTTCCGGTCTGATGAGTCCGTGA SEQ ID NO: 87GGACGAAACAGCAAAAAGAAAAATAAAAA

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. An aptamer that comprises a sequence having atleast 70% identity sequence to FAt8-4 (SEQ ID NO:18), FAt8-11 (SEQ IDNO:20), FAt8-3 (SEQ ID NO:19), FAt6-8 (SEQ ID NO:21), or FAt8-16 (SEQ IDNO:22) and specifically binds to folinic acid, a folate, or a derivativethereof.
 2. An aptamer that specifically binds (6R)-folinic acid andcomprises a sequence having at least 70% identity sequence to FAt8-4(SEQ ID NO:18), FAt8-11 (SEQ ID NO:20), FAt8-3 (SEQ ID NO:19), FAt6-8(SEQ ID NO:21), or FAt8-16 (SEQ ID NO:22).
 3. An aptamer, comprising asequence selected from: a minimally required sequence of FAt8-4 (i) N₁N₂ G N₃ U G C G U G G U A C G U U A U A U U C C G N₄ N₅ (SEQ ID NO:129),where N₁ and N₅ are any complementary pair of nucleotides; N₂ and N₄ areany complementary pair of nucleotides; and N₃ is any nucleotide; aminimally required sequence of FAt8-11 (ii) N₁ N₂ U G C N₃ U G G U A C GU U A U A U U C R G N₄ N₅ (SEQ ID NO:130), where N₁ and N₅ are anycomplementary pair of nucleotides; N₂ and N₄ are any complementary pairof nucleotides; N₃ is any nucleotide; and R is an A or G nucleotide; aminimally required sequence of FAt8-3 (iii) C G U C U G G U C A C G A CC N₁ N₂-N₃ N₄ C C C U C G A A A U C A C G A G G G R G A C R A G A Y (SEQID NO:131), where N₁ and N₄ are any complementary pair of nucleotides;N₂ and N₃ are any complementary pair of nucleotides; R is an A or Gnucleotide; Y is a C or U nucleotide; and the dash indicates anyintervening sequence of nucleotides or two separate nucleotide strands.4. The aptamer of claim 2, wherein the aptamer specifically binds to aligand with a K_(D) of up to 300 nM.
 5. The aptamer of claim 2, operablylinked to an actuator to generate an aptamer-regulated device.
 6. Theaptamer-regulated device of claim 5, wherein the actuator is a ribozyme.7. The aptamer-regulated device of claim 6, wherein the ribozyme is ahammerhead ribozyme.
 8. The aptamer-regulated device of claim 6,comprising a sequence set forth in any of SEQ ID NO:38-43.
 9. Theaptamer-regulated device of claim 5, wherein the actuator is selectedfrom microRNAs, antisense RNAs, RNAi, CRISPR, splicing, small RNAs,ribosome binding sites, internal ribosome entry sites, aptamers, and anycombination thereof.
 10. An aptamer-regulated device, the devicecomprising an aptamer of claim 2, operably linked to an actuator,wherein the actuator is a hammerhead ribozyme and the aptamer and stemIII of the hammerhead ribozyme comprise one or more shared base pairs.11. The aptamer-regulated device of claim 10, wherein theaptamer-regulated device comprises a hammerhead ribozyme interveningsequence of nucleotides within the aptamer sequence.
 12. A DNA sequenceencoding an aptamer-regulated device according to claim
 8. 13. Anaptamer that comprises a sequence having at least 95% identity sequenceto FAt8-4 (SEQ ID NO:18), FAt8-11 (SEQ ID NO:20), FAt8-3 (SEQ ID NO:19),FAt6-8 (SEQ ID NO:21), or FAt8-16 (SEQ ID NO:22) and specifically bindsto folinic acid, a folate, or a derivative thereof.